U.S. patent number 6,921,644 [Application Number 10/101,392] was granted by the patent office on 2005-07-26 for follistatin-3.
This patent grant is currently assigned to Human Genome Sciences, Inc.. Invention is credited to D. Roxanne Duan, Steven M. Ruben.
United States Patent |
6,921,644 |
Duan , et al. |
July 26, 2005 |
**Please see images for:
( Certificate of Correction ) ** |
Follistatin-3
Abstract
The present invention relates to a novel follistatin-3 protein
which is a member of the family of inhibin-related proteins. In
particular, isolated nucleic acid molecules are provided encoding
the human follistatin-3 protein. Follistatin-3 polypeptides are
also provided as are vectors, host cells and recombinant methods
for producing the same. The invention further relates to screening
methods for identifying agonists and antagonists of follistatin-3
activity. Also provided are diagnostic methods for detecting
reproductive system-related disorders and disorders of the
regulation of cell growth and differentiation and therapeutic
methods for treating reproductive system-related disorders and
disorders of the regulation of cell growth and differentiation.
Inventors: |
Duan; D. Roxanne (Bethesda,
MD), Ruben; Steven M. (Olney, MD) |
Assignee: |
Human Genome Sciences, Inc.
(Rockville, MD)
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Family
ID: |
22003166 |
Appl.
No.: |
10/101,392 |
Filed: |
March 20, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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141027 |
Aug 27, 1998 |
6372454 |
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Current U.S.
Class: |
435/7.1; 435/325;
435/326; 435/328; 435/331; 435/335; 530/387.3; 530/387.9;
530/388.1; 530/388.15; 530/388.23; 530/389.2 |
Current CPC
Class: |
A61P
15/00 (20180101); C07K 14/4703 (20130101); A61P
35/00 (20180101); A61K 38/00 (20130101); C07K
2319/00 (20130101); C12N 2799/026 (20130101); A61K
48/00 (20130101); Y02A 50/423 (20180101); C12N
2799/021 (20130101); Y02A 50/30 (20180101); A61K
39/00 (20130101) |
Current International
Class: |
C07K
14/435 (20060101); C07K 14/47 (20060101); A61K
48/00 (20060101); A61K 38/00 (20060101); A61K
39/00 (20060101); C07K 016/24 (); C12N 005/12 ();
G01N 033/53 (); G01N 033/567 () |
Field of
Search: |
;435/7.1,325,326,328,331,335
;530/387.1,387.9,388.1,387.3,388.15,388.23,389.2 ;424/1.49 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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WO 99/25371 |
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May 1999 |
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WO |
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WO 99/31237 |
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Jun 1999 |
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WO |
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WO 99/55865 |
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Nov 1999 |
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WO |
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Primary Examiner: Mertz; Prema
Attorney, Agent or Firm: Human Genome Sciences, Inc.
Parent Case Text
This application is a divisional of U.S. application Ser. No.
09/141,027, filed Aug. 27, 1998 (now U.S. Pat. No. 6,372,454),
which claims benefit under 35 U.S.C .sctn.119(e) of U.S.
Provisional Application Ser. No. 60/056,248, filed Aug. 29, 1997,
which is hereby incorporated by reference in its entirety.
Claims
What is claimed is:
1. An isolated antibody or fragment thereof that specifically binds
to a protein selected from the group consisting of: (a) a protein
whose amino acid sequence consists of amino acid residues -26 to
+237 of SEQ ID NO:2; (b) a protein whose amino acid sequence
consists of amino acid residues +1 to +237 of SEQ ID NO:2; (c) a
protein whose amino acid sequence consists of a portion of SEQ ID
NO:2, wherein said portion is at least 30 contiguous amino acid
residues in length; and (d) a protein whose amino acid sequence
consists of a portion of SEQ ID NO:2, wherein said portion is at
least 50 contiguous amino acid residues in length.
2. The antibody or fragment thereof of claim 1 that specifically
binds protein (a).
3. The antibody or fragment thereof of claim 1 that specifically
binds protein (b).
4. The antibody or fragment thereof of claim 1 that specifically
binds protein (c).
5. The antibody or fragment thereof of claim 1 that specifically
binds protein (d).
6. The antibody or fragment thereof of claim 2 that specifically
binds protein (d).
7. The antibody or fragment thereof of claim 3 wherein said protein
bound by said antibody or fragment thereof is glycosylated.
8. The antibody or fragment thereof of claim 3 wherein said
antibody or fragment thereof is human.
9. The antibody or fragment thereof of claim 3 wherein said
antibody or fragment thereof is polyclonal.
10. The antibody or fragment thereof of claim 3 which is selected
from the group consisting of: (a) a chimeric antibody or fragment
thereof (b) a humanized antibody or fragment thereof; (c) a single
chain antibody; and (d) a Fab fragment.
11. The antibody or fragment thereof of claim 3 which is
labeled.
12. The antibody or fragment thereof of claim 11 wherein the label
is selected from the group consisting of: (a) an enzyme; (b) a
fluorescent label; (c) a luminescent label; and (d) a
bioluminescent label.
13. The antibody or fragment thereof of claim 3 wherein said
antibody or fragment thereof specifically binds to said protein in
a Western blot.
14. The antibody or fragment thereof of claim 3 wherein said
antibody or fragment thereof specifically binds to said protein in
an ELISA.
15. An isolated cell that produces the antibody or fragment thereof
of claim 3.
16. A hybridoma that produces the antibody or fragment thereof of
claim 3.
17. A method of detecting follistatin-3 in a biological sample
comprising: (a) contacting the biological sample with the antibody
or fragment thereof of claim 3; and (b) detecting follistatin-3
protein in the biological sample.
18. The method of claim 17 wherein the antibody or fragment thereof
is polyclonal.
19. An isolated antibody or fragment thereof obtained from an
animal that has been immunized with a protein selected from the
group consisting of: (a) a protein whose amino acid sequence
consists of amino acid residues -26 to +237 of SEQ ID NO:2; (b) a
protein whose amino acid sequence consists of amino acid residues 1
to +237 of SEQ ID NO:2; (c) a protein Comprising the amino acid
sequence of at least 30 contiguous amino acid residues of SEQ ID
NO:2; and (d) a protein comprising the amino acid sequence of at
least 50 contiguous amino acid residues of SEQ ID NO:2; wherein
said antibody or fragment thereof specifically binds to said amino
acid sequence.
20. The antibody or fragment thereof of claim 19 obtained from an
animal immunized with protein (a).
21. The antibody or fragment thereof of claim 19 obtained from an
animal immunized with protein (b).
22. The antibody or fragment thereof of claim 19 obtained from an
animal immunized with protein (c).
23. The antibody or fragment thereof of claim 19 obtained from an
animal immunized with protein (d).
24. The antibody or fragment thereof of claim 19 wherein said
antibody or fragment thereof is monoclonal.
25. The antibody or fragment thereof of claim 19 wherein said
antibody or fragment thereof is polyclonal.
26. The antibody or fragment thereof of claim 19 which is selected
from the group consisting of: (a) a chimeric antibody or fragment
thereof; (b) a humanized antibody or fragment thereof; (c) a single
chain antibody; and (d) a Fab fragment.
27. An isolated monoclonal antibody or fragment thereof that
specifically binds to as protein selected from the group consisting
of: (a) a protein whose amino acid sequence consists of amino acid
residues -26 to +237 of SEQ ID NO:2; (b) a protein whose amino acid
sequence consists of amino acid residues +1 to +237 of SEQ ID NO:2;
(c) a protein whose amino acid sequence consists of a portion of
SEQ ID NO:2, wherein said portion is at least 30 contiguous is
amino acid residues in length; and (d) a protein whose amino acid
sequence consists of a portion of SEQ ID NO:2, wherein said portion
is at least 50 contiguous is amino acid residues in length.
28. The antibody or fragment thereof of claim 27 that specifically
binds protein (a).
29. The antibody or fragment thereof of claim 27 that specifically
binds protean (b).
30. The antibody or fragment thereof of claim 27 that specifically
binds protein (c).
31. The antibody or fragment thereof of claim 27 that specifically
binds protein (d).
32. The antibody or fragment thereof of claim 28 that specifically
binds protein (c).
33. The antibody or fragment thereof of claim 29 that wherein said
protein bound by said antibody or fragment thereof is
glycosylated.
34. The antibody or fragment thereof of claim 29 that wherein said
antibody or fragment thereof is human.
35. The antibody or fragment thereof of claim 29 that which is
selected from the group consisting of: (a) a chimeric antibody or
fragment thereof; (b) a humanized antibody or fragment thereof; (c)
a single chain antibody: and (d) a Fab fragment.
36. The antibody or fragment thereof of claim 29 which is
labeled.
37. The antibody or fragment thereof of claim 36 wherein the label
is selected from the group consisting of: (a) an enzyme; (b) a
fluorescent label; (c) a luminescent label; and (d) a
bioluminescent label.
38. The antibody or fragment thereof of claim 29 wherein said
antibody of fragment thereof specifically binds to said protein in
a Western blot.
39. The antibody or fragment thereof of claim 29 wherein said
antibody or fragment thereof specifically binds to said protein in
an ELISA.
40. An isolated cell that produces the antibody or fragment thereof
of claim 29.
41. A hybridoma that produces the antibody or fragment thereof of
claim 29.
42. A method of detecting follistatin-3 protein in a biological
sample comprising: (a) contacting the biological sample with the
antibody or fragment thereof if claim 29; and (b) detecting
follistatin-3 protein in the biological sample.
43. An isolated antibody or fragment thereof that specifically
binds to a protein selected from the group consisting of: (a) a
protein whose amino acid sequence consists of the amino acid
sequence of the full-length polypeptide encoded by the cDNA
contained in ATCC.RTM. Deposit Number 209199; (b) a protein whose
amino acid sequence consists of the amino acid sequence of the
mature form of the polypeptide encoded by the cDNA contained in
ATCC.RTM. Deposit Number 209199; (c) a protein whose amino acid
sequence consists of the amino acid sequence of a portion of the
polypeptide encoded by the cDNA contained in ATCC.RTM. Deposit
Number 209199, wherein said portion is at least 30 contiguous amino
acid residues in length; and (d) a protein whose amino acid
sequence Consists of the amino acid sequence of a portion of the
polypeptide encoded by the cDNA contained in ATCC.RTM. Deposit
Number 209199, wherein said portion is at least 50 contiguous amino
acid residues in length.
44. The antibody or fragment thereof of claim 43 that specifically
binds protein (a).
45. The antibody or fragment thereof of claim 43 that specifically
binds protein (b).
46. The antibody or fragment thereof of claim 43 that specifically
binds protein (c).
47. The antibody or fragment thereof of claim 43 that specifically
binds protein (d).
48. The antibody or fragment thereof of claim 44 that specifically
binds protein (c).
49. The antibody or fragment thereof of claim 45 that wherein said
protein bound by said antibody or fragment thereof is
glycosylated.
50. The antibody or fragment thereof of claim 45 that wherein said
antibody or fragment thereof is human.
51. The antibody or fragment thereof of claim 45 wherein said
antibody or fragment thereof is polyclonal.
52. The antibody or fragment thereof of claim 45 which is selected
from the group consisting of: (a) a chimeric antibody or fragment
thereof; (b) a humanized antibody or fragment thereof; (c) a single
chain antibody; and (d) a Fab fragment.
53. The antibody or fragment thereof of claim 45 which is
labeled.
54. The antibody or fragment thereof of claim 45 wherein the label
is selected from the group consisting of: (a) an enzyme; (b) a
fluorescent label; (c) a luminescent label; and (d) a
bioluminescent label.
55. The antibody or fragment thereof of claim 45 wherein said
antibody or fragment thereof specifically binds to said protein in
a Western blot.
56. The antibody or fragment thereof of claim 45 wherein said
antibody or fragment thereof specifically binds to said protein in
an ELISA.
57. An isolated cell that produces the antibody or fragment thereof
of claim 45.
58. A hybridoma that produces the antibody or fragment thereof of
claim 45.
59. A method of detecting follistatin-3 protein in a biological
sample comprising: (a) contacting the biological sample with the
antibody or fragment thereof of claim 45; and (b) detecting
follistatin-3 protein in the biological sample.
60. The method of claim 59 wherein the antibody or fragment thereof
is polyclonal.
61. An isolated antibody or fragment thereof obtained from an
animal that has been immunized with a protein selected from the
group consisting of: (a) a protein comprising the amino acid
sequence of the full-length polypeptide encoded by the cDNA
contained in ATCC.RTM. Deposit Number 209199; (b) a protein
comprising the amino acid sequence of the mature form of the
polypeptide encoded by the cDNA contained in ATCC.RTM. Deposit
Number 209199; (c) a protein comprising the amino acid sequence of
at least 30 contiguous amino acid residues of the polypeptide
encoded by the cDNA contained in ATCC.RTM. Deposit Number 209199;
and (d) a protein comprising the amino acid sequence of at least 50
contiguous amino acid residues the polypeptide encoded by the cDNA
contained in ATCC.RTM. Deposit Number 209199; wherein said antibody
or fragment thereof specifically binds to said amino acid
sequence.
62. The antibody or fragment thereof of claim 61 obtained from an
animal immunized with protein (a).
63. The antibody or fragment thereof of claim 61 obtained from an
animal immunized with protein (b).
64. The antibody or fragment thereof of claim 61 obtained from an
animal immunized with protein (c).
65. The antibody or fragment thereof of claim 61 obtained from an
animal immunized with protein (d).
66. The antibody or fragment thereof of claim 61 wherein the
antibody or fragment thereof is monoclonal.
67. The antibody or fragment thereof of claim 61 wherein the
antibody or fragment thereof is polyclonal.
68. The antibody or fragment thereof of claim 61 which is selected
from the group consisting of: (a) a chimeric antibody or fragment
thereof; (b) a humanized antibody or fragment thereof; (c) a single
chain antibody; and (d) a Fab fragment.
69. An isolated monoclonal antibody or fragment thereof that
specifically binds to a protein selected from the group consisting
of: (a) a protein whose amino acid sequence consists of the amino
acid sequence of the full-length polypeptide encoded by the cDNA
contained in ATCC.RTM. Deposit Number 209199; (b) a protein whose
amino acid sequence consists of the amino acid sequence of the
mature form of the polypeptide encoded by the cDNA contained in
ATCC.RTM. Deposit Number 209199; (c) a protein whose amino acid
sequence consists of the amino acid sequence of a portion of the
polypeptide encoded by the cDNA contained in ATCC.RTM. Deposit
Number 209199, wherein said portion is at least 30 contiguous amino
acid residues in length; and (d) a protein whose amino acid
sequence consists of the amino acid sequence of a portion of the
polypeptide encoded by the cDNA contained in ATCC.RTM. Deposit
Number 209199, wherein said portion is at least 50 contiguous amino
acid residues in length.
70. The antibody or fragment thereof of claim 69 that specifically
binds protein (a).
71. The antibody or fragment thereof of claim 69 that specifically
binds protein (b).
72. The antibody or fragment thereof of claim 69 that specifically
binds protein (c).
73. The antibody or fragment thereof of claim 69 that specifically
binds protein (d).
74. The antibody or fragment thereof of claim 70 that specifically
binds protein (c).
75. The antibody or fragment thereof of claim 71 that wherein said
protein bound by said antibody or fragment thereof is
glycosylated.
76. The antibody or fragment thereof of claim 71 that wherein said
antibody or fragment thereof is human.
77. The antibody or fragment thereof of claim 71 that which is
selected from the group consisting of: (a) a chimeric antibody or
fragment thereof; (b) a humanized antibody or fragment thereof; (c)
a single chain antibody; and (d) a Fab fragment.
78. The antibody or fragment thereof of claim 71 which is
labeled.
79. The antibody or fragment thereof of claim 78 wherein the label
is selected from the group consisting of: (a) an enzyme; (b) a
fluorescent label; (c) a luminescent label; and (d) a
bioluminescent label.
80. The antibody or fragment thereof of claim 71 wherein said
antibody or fragment thereof specifically binds to said protein in
a Western blot.
81. The antibody or fragment thereof of claim 71 wherein said
antibody or fragment thereof specifically binds to said protein in
an ELISA.
82. An isolated cell that produces the antibody or fragment thereof
of claim 71.
83. A hybridoma that produces the antibody or fragment thereof of
claim 71.
84. A method of detecting follistatin-3 protein in a biological
sample comprising: (a) contacting the biological sample with the
antibody or fragment thereof of claim 71; and (b) detecting the
follistatin-3 protein in the biological sample.
85. An isolated antibody or fragment thereof that specifically
binds follistatin-3 protein purified from a cell culture wherein
said follistatin-3 protein is encoded by a polynucleotide encoding
amino acid -26 to +237 of SEQ ID NO:2.
86. The antibody or fragment thereof of claim 85 wherein said
antibody or fragment thereof is monoclonal.
87. The antibody or fragment thereof of claim 85 wherein said
antibody or fragment thereof is polyclonal.
88. The antibody or fragment thereof of claim 85 wherein said
antibody or fragment thereof is human.
89. The antibody or fragment thereof of claim 85 which is selected
from the group consisting of: (a) chimeric antibody or fragment
thereof; (b) a humanized antibody or fragment thereof; (c) a single
chain antibody; and (d) a Fab fragment.
90. The antibody or fragment thereof of claim 85 wherein said
antibody or fragment thereof specifically binds to said protein in
a Western blot.
91. The antibody or fragment thereof of claim 85 wherein said
antibody or fragment thereof specifically binds to said protein in
an ELISA.
Description
FIELD OF THE INVENTION
The present invention relates to a novel human gene encoding a
polypeptide which is a member of the family of inhibin-related
proteins. More specifically, isolated nucleic acid molecules are
provided encoding a human polypeptide named follistatin-3.
Follistatin-3 polypeptides are also provided, as are vectors, host
cells and recombinant methods for producing the same. Also provided
are diagnostic methods for detecting disorders related to the
reproductive system, and therapeutic methods for treating such
disorders. The invention further relates to screening methods for
identifying agonists and antagonists of follistatin-3 activity.
BACKGROUND OF THE INVENTION
The family of inhibin-related proteins currently consists of at
least four groups of members: inhibins, activins, and two splice
variants of follistatin-1 (315 and 288 amino acids). Inhibins and
activins are members of the transforming growth factor (TGF)-.beta.
superfamily and function with opposing actions in a variety of
capacities in paracrine and autocrine regulation of
both-reproductive and nonreproductive organs including the liver,
kidney, adrenal glands, bone marrow, placenta, anterior pituitary,
and brain (Ying, S. Y., et al., Proc. Soc. Exp. Biol. Med.
214:114-122 (1997); Mather, J. P., et al., Proc. Soc. Exp. Biol.
Med. 215:209-222 (1997)). Although the follistatins are not closely
related to the TGF-.beta. family, they still play a major role in
the follical stimulating hormone (FSH) synthetic pathway by
increasing estradiol production and by functioning directly as high
affinity activin-binding proteins. Inhibins, activins, and
follistatin-1 were all initially identified as regulators of
pituitary FSH secretion, but have more recently been further
characterized to function as growth factors, embryo modulators, and
immune factors (Petraglia, F. Placenta 18:3-8 (1997)). In addition,
each of these factors is involved with the regulation of
gonadotropin biosynthesis and secretion, ovarian and placental
steroidogenesis, and oocyte and spermatogonial maturation
(Halvorson, L. M. and DeCherney, A. H. Fertil. Steril. 65:459-469
(1996)).
FSH is a vital component of the regulatory cascade governing
development of human oocytes. Primary oocytes in newborns are
arrested in the prophase stage of Meiosis I and are surrounded by a
1-2 cell thick layer of follicle cells constituting a structure
termed the primordial follicle. In concert with other factors,
stimulation of the primordial follicle with FSH initiates its
progression to the more complex structures designated the
developing and antral follicles (Ueno, N., et al., Proc. Natl.
Acad. Sci. USA 84:8282-8286 (1987); Robertson, D. M., et al.,
Biochem. Biophys. Res. Comm. 149:744-749 (1987)). The antral
follicle consists of an enlarged oocyte surrounded by an increased
number of follicle cells, a zona pellucida, cortical granules, and
a fluid-filled cavity termed the antrum. It is in this state that
thousands of developing oocytes are maintained until puberty. Each
month following this point, a surge in the local concentration of
several additional hormones and other factors, primarily
leuteinizing hormone (LH), stimulates accelerates the growth of
roughly 15-20 of the developing follicles in the ovary. Only one of
these structures will ultimately complete the developmental
progression of its enclosed oocyte to the metaphase stage of
Meiosis II. The single stimulated follicle will then continue to
enlarge until it bursts at the surface of the ovary and releases
the oocyte, still surrounded with a coating of follicle cells, for
potential fertilization (Boruslaeger, E. A., et al., Dev. Biol.
114:453-462 (1986); Masui, Y. and Clarke, H. J. Int. Rev. Cytol.
57:185-282 (1979); Richards, J. S. Recent Prog. Horm. Res.
35:343-373 (1979)).
Follistatin also plays a central role in the above-described
process of follicle development. Follistatin binds
stoichiometrically to activins and, as a result, inhibits the
activin-induced augmentation of FSH-release from cultured pituitary
cells (Kogawa, K., et al., Endocrinology 128:1434-1440 (1991)).
Further evidencing a feedback mechanism, cultured granulosa cells
produce and secrete follistatin in response to treatment with FSH
(Saito, S., et al., Biochem. Biophys. Res. Comm. 176:413-422
(1991); Klein, R., et al., Endocrinology 128:1048-1056 (1991)).
Furthermore, it has been determined by synthesizing the results of
a number of studies, that follistatin, activin, FSH, LH, and other
factors function in concert in a variety of interrelated mechanisms
to regulate many developmental processes, including the development
of follicles. For example, in the presence of FSH, activin can
augment both LH receptor expression and progesterone production by
rat granulosa cells (Sugino, H., et al., Biochem. Biophys. Res.
Comm. 153:281-288 (1988)). In addition, activin can significantly
enhance the ability of granulosa cells to express FSH receptor and
produce inhibin even in the absence of FSH (Nakamura, T., et al.,
Biochim. Biophys. Acta 1135:103-109 (1992); Sugino, H., et al.,
supra; Hasegawa, Y., et al., Biochem. Biophys. Res. Comm.
156:668-674 (1988)). These and other studies provide support for
the idea that follistatin and activin play important roles in the
regulation of granulosa cellular differentiation.
In addition to the many well-characterized effects which
follistatin, activin, and inhibin elicit on the regulation of
various developmental processes in the reproductive system, a large
number of studies have more recently begun to define regulatory
roles for these molecules in a variety of other tissues and
systems. For example, during early embryonic development in
Xenoptus laevis, the action of activin A in developing targets of
ciliary ganglion neurons is regulated by localized expression of
follistatin (Hemmati-Brivanlou, A. and Melton, D. A. Nature
359:609-614 (1992); Hemmati-Brivanlou, A. and Melton, D. A. Cell
77:273-281 (1994)). In addition, overexpression of follistatin
leads to induction of neural tissue (Hemmati-Brivanlou, A., et al.,
Cell 77:283-295 (1994)). In the mouse, follistatin mRNA is first
detected on embryonic day 5.5 in the deciduum, and, subsequently,
in the developing hindbrain, somites, vibrissae, teeth, epidermis,
and muscle (van den Eihnden-van Raaij, A. J. M., et al., Dev. Biol.
154:356-365 (1992); Albano, R. M., et al., Development 120:803-813
(1994); Feijen, A., et al., Development 120:3621-3637 (1994)).
Evidence of the relative importance of such a varied expression of
follistatin is provided by Matzuk and colleagues (Nature
374:360-363 (1995)) who demonstrate that follistatin-deficient mice
are retarded in their growth, have decreased mass of the diaphragm
and intercostal muscles, shiny taut skin, skeletal defects of the
hard palate and the thirteenth pair of fibs, their whisker and
tooth development is abnormal, they fail to breathe, and die within
hours of birth. Since the defects in mice deficient in follistatin
are far more widespread than in mice deficient in activin, Matzuk
and coworkers (supra) suggest that follistatin may modulate the
cell growth and differentiation regulatory actions of additional
members of the TGF-.beta. superfamily.
Thus, there is a need for polypeptides that function as regulators
of reproductive development, embryonic development, and cell growth
and differentiation since disturbances of such regulation may be
involved in disorders relating to reproduction and the regulation
of cell growth and differentiation. Therefore, there is a need for
identification and characterization of such human polypeptides
which can play a role in detecting, preventing, ameliorating or
correcting such disorders.
SUMMARY OF THE INVENTION
The present invention provides isolated nucleic acid molecules
comprising a polynucleotide encoding at least a portion of the
follistatin-3 polypeptide having the complete amino acid sequence
shown in SEQ ID NO:2 or the complete amino acid sequence encoded by
the cDNA clone deposited as plasmid DNA as ATCC.RTM. Deposit Number
209197 on Aug. 8, 1997. The nucleotide sequence determined by
sequencing the deposited follistatin-3 clone, which is shown in
FIGS. 1A, 1B, and 1C (SEQ ID NO:1), contains a open reading frame
encoding a complete polypeptide of 263 amino acid residues,
including an initiation codon encoding an N-terminal methionine at
nucleotide positions 19-21, and a predicted molecular weight of
about 27.7 kDa. Nucleic acid molecules of the invention include
those encoding the complete amino acid sequence excepting the
N-terminal methionine shown in SEQ ID NO:2, or the complete amino
acid sequence excepting the N-terminal methionine encoded by the
cDNA clone in ATCC.RTM. Deposit Number 209197, which molecule also
can encode additional amino acids fused to the N-terminus of the
follistatin-3 amino acid sequence.
The encoded polypeptide has a predicted leader sequence of 26 amino
acids underlined in FIG. 1A; and the amino acid sequence of the
predicted mature follistatin-3 protein is also shown in FIGS. 1A,
1B, and 1C, as amino acid residues 27-263 and as residues 1-237 in
SEQ ID NO:2.
Thus, one aspect of the invention provides an isolated nucleic acid
molecule comprising a polynucleotide comprising a nucleotide
sequence selected from the group consisting of: (a) a nucleotide
sequence encoding the follistatin-3 polypeptide having the complete
amino acid Sequence in SEQ ID NO:2 (i.e., positions -26 to 237 of
SEQ ID NO.2), (b) a nucleotide sequence encoding the follistatin-3
polypeptide having the complete amino acid sequence in SEQ ID NO:2
excepting the N-terminal methionine (i.e., positions -25 to 237 of
SEQ ID NO:2); (c) a nucleotide sequence encoding the predicted
mature follistatin-3 polypeptide having the amino acid sequence at
positions 1 to 237 in SEQ ID NO:2; (d) a nucleotide sequence
encoding the follistatin-3 polypeptide having the complete ammo
acid sequence encoded by the cDNA clone contained in ATCC.RTM.
Deposit No. 209197; (e) a nucleotide sequence encoding the
follistatin-3 polypeptide having the complete amino acid sequence
excepting the N-terminal methionine encoded by the cDNA clone
contained in ATCC.RTM. Deposit No. 209197; (I a nucleotide sequence
encoding the mature follistatin-3 polypeptide having the amino acid
sequence encoded by the cDNA clone contained in ATCC.RTM. Deposit
No. 209197; and (g) a nucleotide sequence complementary to any of
the nucleotide Sequences in (a), (b), (c), (d), (e) or (f)
above.
Further embodiments of the invention include isolated nucleic acid
molecules that comprise a polynucleotide having a nucleotide
sequence at least 90% identical, and more preferably at least 95%,
96%, 97%, 98% or 99% identical, to any of the nucleotide sequences
in (a), (b), (c), (d), (e), .(f) or (g), above, or a polynucleotide
which hybridizes under stringent hybridization conditions to a
polynucleotide in (a), (b), (c), (d), (e), (f) or (g), above. This
polynucleotide which hybridizes does not hybridize under stringent
hybridization conditions to a polynucleotide having a nucleotide
sequence consisting of only A residues or of only T residues.
An additional nucleic acid embodiment of the invention relates to
an isolated nucleic acid molecule comprising a polynucleotide which
encodes the amino acid sequence of an epitope-bearing portion of a
follistatin-3 polypeptide having an amino acid sequence in (a),
(b), (c), (d), (e) or (f), above. A further embodiment of the
invention relates to an isolated nucleic acid molecule comprising a
polynucleotide which encodes the amino acid sequence of a
follistatin-3 polypeptide having an amino acid sequence which
contains at least one amino acid substitution, but not more than 50
amino acid substitutions, even more preferably, not more than 40
amino acid substitutions, still more preferably, not more than 30
amino acid substitutions, and still even more preferably, not more
than 20 amino acid substitutions. Of course, in order of
ever-increasing preference, it is highly preferable for a
polynucleotide which encodes the amino acid sequence of a
follistatin-3 polypeptide to have an amino acid sequence which
contains not more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid
substitutions. Conservative substitutions are preferable.
The present invention also relates to recombinant vectors, which
include the isolated nucleic acid molecules of the present
invention; and to host cells containing the recombinant vectors, as
well as to methods of making such vectors and host cells and for
using them for production of follistatin-3 polypeptides or peptides
by recombinant techniques.
In accordance with a further aspect of the present invention, there
is provided a process for producing such polypeptide by recombinant
techniques comprising culturing recombinant prokaryotic and/or
eukaryotic host cells, containing a follistatin-3 nucleic acid
sequence, under conditions promoting expression of said protein and
subsequent recovery of said protein.
The invention further provides in isolated follistatin-3
polypeptide comprising: an amino acid sequence selected from the
group consisting of: (a) tie amino acid sequence of the full-length
follistatin-3 polypeptide having the complete amino acid sequence
shown in SEQ ID NO:2 (i.e., positions -26 to 237 of SEQ ID NO:2);
(b) the amino acid sequence of the full-length follistatin-3
polypeptide having the complete amino acid sequence shown in SEQ ID
NO:2 excepting the N-terminal methionine (i.e., positions -25 to
237 of SEQ ID NO:2); (c) the amino acid sequence of the predicted
mature follistatin-3 polypeptide having the amino acid sequence at
positions 1 to 237 in SEQ ID NO:2; (d) the amino acid sequence of
the full-length follistatin-3 polypeptide having the complete amino
acid sequence encoded by the cDNA clone contained in ATCC.RTM.
Deposit No. 209197; (e) the amino acid sequence of the full-length
follistatin-3 polypeptide having the complete amino acid sequence
excepting the N-terminal methionine encoded by the cDNA clone
contained in ATCC.RTM. Deposit No. 209197; and (f) the amino acid
sequence of the mature follistatin-3 polypeptide having the amino
acid sequence encoded by the cDNA clone contained in ATCC.RTM.
Deposit No.209197. The polypeptides of the present invention also
include polypeptides having an amino acid sequence at least 80%
identical, more preferably at least 90% identical, and still more
preferably 95%, 96%, 97%, 98% or 99% identical to those described
in (a), (b), (c), (f), (e) or (0 above, as well as polypeptides
having an amino acid sequence with at least 90% similarity, and
more preferably at least 95% similarity, to those above.
An additional embodiment of this aspect of the invention relates to
a peptide or polypeptide which comprises the amino acid sequence of
an epitope-bearing portion of a follistatin-3 polypeptide having an
amino acid sequence described in (a), (b), (c), (d), (e) or (f)
above. Peptides or polypeptides having the amino acid sequence of
an epitope-bearing portion of a follistatin-3 polypeptide of the
invention include portions of such polypeptides with at least six
or seven, preferably at least nine, and more preferably at least
about 30 amino acids to about 50 amino acids, although
epitope-bearing polypeptides of any length up to and including the
entire amino acid sequence of a polypeptide of the invention
described above also are included in the invention.
A further embodiment of the invention relates to a polypeptide
which comprises the amino acid sequence of a follistatin-3
polypeptide having an amino acid sequence which contains at least
one amino acid substitution, but not more than 50 amino acid
substitutions, even more preferably, not more than 40 amino acid
substitutions, still more preferably, not more than 30 amino acid
substitutions, and still even more preferably, not more than 20
amino acid substitutions. Of course, in order of ever-increasing
preference, it is highly preferable for a peptide or polypeptide to
have an amino acid sequence which comprises the amino acid sequence
of a follistatin-3 polypeptide, which contains at least one, but
not more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid
substitutions. In specific embodiments, the number of additions,
substitutions, and/or deletions in the amino acid sequence of FIGS.
1A, 1B, and 1C, or fragments thereof (e.g., the mature form and/or
other fragments described herein), is 1-5, 5-10, 5-25, 5-50, 10-50
or 50-150, conservative amino acid substitutions are
preferable.
In another embodiment, the invention provides an isolated antibody
that binds specifically to a follistatin-3 polypeptide having an
amino acid sequence described in (a), (b), (c), (d), (e) or (f)
above. The invention further provides methods for isolating
antibodies that bind specifically to a follistatin-3 polypeptide
having an amino acid sequence as described herein. Such antibodies
are useful diagnostically or therapeutically as described
below.
The invention also provides for pharmaceutical compositions
comprising follistatin-3 polypeptides, particularly human
follistatin-3 polypeptides, which may be employed, for instance, to
treat cancers and other cellular growth and differentiation
disorders, as well as disorders of the reproductive system. Methods
of treating individuals in need of follistatin-3 polypeptides are
also provided.
The invention further provides compositions comprising a
follistatin-3 polynucleotide or a follistatin-3 polypeptide for
administration to cells in vitro, to cells ex vivo and to cells in
vivo, or to a multicellular organism. In certain particularly
preferred embodiments of this aspect of the invention, the
compositions comprise a follistatin-3 polynucleotide for expression
of a follistatin-3 polypeptide in a host organism for treatment of
disease. Particularly preferred in this regard is expression in a
human patient for treatment of a dysfunction associated with
aberrant endogenous activity of follistatin-3.
The present invention also provides a screening method for
identifying compounds capable of enhancing or inhibiting a
biological activity of the follistatin-3 polypeptide, which
involves contacting a ligand which is inhibited by the
follistatin-3 polypeptide with the candidate compound in the
presence of a follistatin-3 polypeptide, assaying receptor-binding
activity of the ligand in the presence of the candidate compound
and of follistatin-3 polypeptide, and comparing the ligand activity
to a standard level of activity, the standard being assayed when
contact is made between the ligand itself in the presence of the
follistatin-3 polypeptide and the absence of the candidate compound
In this assay, an increase in ligand activity over the standard
indicates that the candidate compound is an agonists of
follistatin-3 activity and a decrease in ligand activity compared
to the standard indicates that the compound is an antagonist of
follistatin-3 activity.
In another aspect, a screening assay for agonists and antagonists
is provided which involves determining the effect a candidate
compound has on follistatin-3 binding to activin or an activin-like
molecule. In particular, the method involves contacting the activin
or an activin-like molecule with a follistatin-3 polypeptide and a
candidate compound and determining whether follistatin-3
polypeptide binding to the activin or an activin-like molecule is
increased or decreased due to the presence of the candidate
compound. In this assay, an increase in binding of follistatin-3
over the standard binding indicates that the candidate compound is
an agonists of follistatin-3 binding activity and a decrease in
follistatin-3 binding compared to the standard indicates that the
compound is an antagonist of follistatin-3 binding activity.
It has been discovered that follistatin-3 is expressed not only in
Hodgkin's Lymphoma but also in synovial fibroblasts, gall bladder,
resting and serum-induced smooth muscle, testes, Merkel cells, HEL
cells, hippocampus, TNF-.alpha.- and IFN-induced epithelial cells,
keratinocyte, amygdala depression, HL-60 cells, hepatoma,
progesterone-treated epidermal cells, endothelial cells, HSC172
cells, epithelioid sarcoma, activated T-cells, breast lymph node,
pancreatic carcinoma, fetal dura mater, fetal lung, epididymis,
placenta, dendritic cells, rejected kidney, and uterine cancer.
Therefore, nucleic acids of the invention are useful as
hybridization probes for differential identification of the
tissue(s) or cell type(s) present in a biological sample.
Similarly, polypeptides and antibodies directed to those
polypeptides are useful to provide immunological probes for
differential identification of the tissue(s) or cell type(s). In
addition, for a number of disorders of the above tissues or cells,
particularly of the reproductive system, or disorders of the
regulation of cell growth and differentiation, significantly higher
or lower levels of follistatin-3 gene expression may be detected in
certain tissues (e.g., cancerous and wounded tissues) or bodily
fluids (e.g., serum, plasma, urine, synovial fluid or spinal fluid)
taken from an individual having such a disorder, relative to a
"standard" follistatin-3 gene expression level, i.e., the
follistatin-3 expression level in healthy tissue from an individual
not having the reproductive system or regulation of cell growth and
differentiation disorder. Thus, the invention provides a diagnostic
method useful during diagnosis of such a disorder, which involves:
(a) assaying follistatin-3 gene expression level in cells or body
fluid of an individual; (b) comparing the follistatin-3 gene
expression level with a standard follistatin-3 gene expression
level, whereby an increase or decrease in the assayed follistatin-3
gene expression level compared to the standard expression level is
indicative of disorder in the reproductive system or of a disorder
of the regulation of cell growth and differentiation.
An additional aspect of the invention is related to a method for
treating an individual in need of an increased level of
follistatin-3 activity in the body comprising administering to such
an individual a composition comprising a therapeutically effective
amount of an isolated follistatin-3 polypeptide of the invention or
an agonists thereof.
A still further aspect of the invention is related to a method for
treating an individual in need of a decreased level of
follistatin-3 activity in the body comprising, administering to
such an individual a composition comprising a therapeutically
effective amount of an follistatin-3 antagonist. Preferred
antagonists for use in the present invention are
follistatin-3-specific antibodies.
BRIEF DESCRIPTION OF THE FIGURES
FIGS. 1A, 1B, and 1C show the nucleotide sequence (SEQ ID NO:1) and
deduced amino acid sequence (SEQ ID NO:2) of follistatin-3.
The predicted leader sequence of about 26 amino acids is
underlined. Note that the methionine residue at the beginning of
the leader sequence in FIG. 1A is shown in position number
(positive) 1, whereas the leader positions in the corresponding
sequence of SEQ ID NO:2 are designated with negative position
numbers. Thus, the leader sequence positions 1 to 26 in FIG. 1A
correspond to positions -26 to -1 in SEQ ID NO:2.
Two potential asparagine-linked glycosylation sites are marked in
the amino acid sequence of follistatin-3. The sites are
asparagine-73 and asparagine-215 in FIG. 1A (asparagine-47 and
asparagine-179 in SEQ ID NO:2), and are with the bold pound symbol
(#) above the nucleotide sequence coupled with a bolded one letter
abbreviation for the asparagine (N) in the amino acid sequence in
FIG. 1A; that is, the actual asparagine residues which are
potentially glycosylated is bolded in FIG. 1A. The potential
N-linked glycosylation sequences are found at the following
locations in the follistatin-3 amino acid sequence: N-73 through
H-76 (N-73, L-74, T-75, H-76) and N-215 through Y-218 (N-215,
V-216, T-217, Y-218). A potential Protein Kinase C (PKC)
phosphorylation site is also marked in FIG. 1A with a bolded
tyrosine symbol (T) in the follistatin-3 amino acid sequence and an
asterisk (*) above the first nucleotide encoding that tyrosine
residue in the follistatin-3 nucleotide sequence. The potential PKC
phosphorylation sequence is found in the follistatin-3 amino acid
sequence from residue T-141 through residue R-143 (T-141, Y-142,
R-143). Potential Casein Kinase II (CK2) phosphorylation sites are
also marked in FIG. 1A with a bolded tyrosine or serine symbol (T
or S) in the follistatin-3 amino acid sequence and an asterisk (*)
above the first nucleotide encoding the appropriate tyrosine or
serine residue in the follistatin-3 nucleotide sequence. Potential
CK2 phosphorylation sequences are found at the following locations
in the follistatin-3 amino acid sequence: T-57 through E-60 (T-57,
R-58, A-59, E-60); T-141 through D-144 (T-141, Y-142, R-143,
D-144); T-246 through E-249 (T-246, P-247, E-248, E-249); and S-255
through E-258 (S-255, A-256, E-257, E-258). Ten potential
myristylation sites are found in the follistatin-3 amino acid
sequence shown in FIG. 1A. Potential myristylation sites are marked
in FIG. 1A with a double underline delineating the amino acid
residues representing each potential myristolation site in the
follistatin-3 amino acid sequence. The potential myristolation
sites are located in the following positions in the follistatin-3
amino acid sequence: G-43 through C-48 (G-43, Q-44, E-45, A-46,
T-47, C-48); G-65 through A-70 (G-65, N-66, I-67, D-68, T-69,
A-70); G-78 through L-83 (G-78, N-79, K-80, 1-81, N-82, L-83); G-88
through L-93 (G-88, L-89, V-90, H-91, C-92, L-93); G-136 through
T-141 (G-136, S-137, D-138, G-139, A-140, T-141); G-188 through
V-193 (G-188, S-189, A-190, H-191, C-192, V-193); G-207 through
G-212 (G-207, Q-208, E-209, L-210, C-211, G-212); G-236 through
G-241 (G-236, V-237, R-238, H-239, A-240, G-241); G-241 through
T-246 (G-241, S-242, C-243, A-244, G-245, T-246); and G-252 through
E-257 (G-252, G-253, E-254, S-255, A-256, E-257).
FIG. 2 shows the regions of identity between the amino acid
sequences of the follistatin-3 protein and translation product of
the human mRNA for follistatin-1 (SEQ ID NO:3), determined by the
computer program Bestfit (Wisconsin Sequence Analysis Package,
Version 8 for Unix, Genetics Computer Group, University Research
Park, 575 Science Drive, Madison, Wis. 53711) using the default
parameters.
FIG. 3 shows an analysis of the follistatin-3 amino acid sequence
(SEQ ID NO:2). Alpha, beta, turn and coil regions; hydrophilicity
and hydrophobicity; amphipathic regions; flexible regions;
antigenic index and surface probability, as predicted using default
parameters of the recited computer programs, are shown.
In the "Antigenic Index or Jameson-Wolf" graph, the positive peaks
indicate locations of the highly antigenic regions of the
follistatin-3 protein, i.e., regions from which epitope-bearing
peptides of the invention can be obtained. Non-limiting examples of
antigenic polypeptides or peptides that can be used to generate
follistatin-3-specific antibodies include: a polypeptide comprising
amino acid residues Lys-54 to Asp-62, Val-91 to Leu-99, Lys-100 to
Gln-108, Cys-116 to Pro-124, Gln-140 to Leu-148, Trp-156 to
Ser-164, Arg-170 to Gln-181, Cys-212 to Phe-224, Tyr-239 to
Thr-247, Pro-251 to Met-259, and Asp-263, to His-271 of SEQ ID
NO:2.
The data presented in FIG. 3 are also represented in tabular form
in Table I. The columns are labeled with the headings "Res",
"Position", and Roman Numerals I-XIV. The column headings refer to
the following features of the amino acid sequence presented in FIG.
3 and Table I: "Res": amino acid residue of SEQ ID NO:2 or FIG. 1A
(which is the identical sequence shown in SEQ ID NO:2, with the
exception that the residues are numbered 1-263 in FIG. 1A and -18
through 348 in SEQ ID NO:4); "Position": position of the
corresponding residue within SEQ ID NO:2 or FIGS. 2A and 2B (which
is the identical sequence shown in SEQ ID NO:4, with the exception
that the residues are numbered 1-366 in FIGS. 2A and 2B and -18
through 348 in SEQ ID NO:4); I: Alpha, Regions--Gamier-Robson; II:
Alpha, Regions--Chou-Fasman; III: Beta, Regions--Garnier-Robson;
IV: Beta, Regions--Chou-Fasman; V: Turn, Regions-Garnier-Robson;
VI: Turn, Regions--Chou-Fasman; VII: Coil, Regions--Garnier-Robson;
VIII: Hydrophilicity Plot--Kyte-Doolittle; IX: Hydrophobicity
Plot-Hopp-Woods; X: Alpha, Amphipathic Regions--Eisenberg; XI:
Beta, Amphipathic Regions--Eisenberg; XII: Flexible
Regions--Karplus-Schulz; XIII: Antigenic Index-Jameson-Wolf; and
XIV: Surface Probability Plot-Emini.
DETAILED DESCRIPTION
The present invention provides isolated nucleic acid molecules
comprising a polynucleoticle encoding a folliStatin-3 polypeptide
having the amino acid sequence shown in SEQ ID NO:2, which was
determined by sequencing a cloned cDNA. The nucleotide sequence
shown in FIGS. 1A. 1B, and 1C (SEQ ID NO:1) was obtained by
sequencing the HDTAH85 clone, which was deposited On Aug. 2, 1997
at the American Type Culture Collection, 10801 University
floulevard, Manassas, Va. 20110-2209, and given accession number
ATCC.RTM. 209197. The deposared clone is conTained in the
pBluescript SK(-) plasmid (Stratagene, La Jolla, Calif.).
Nucleic Acid Molecules
Unless otherwise indicated, all nucleotide sequences determined by
sequencing a DNA molecule herein were determined using an automated
DNA sequencer (such as the Model 373 from Applied Biosystems, Inc.,
Foster City, Calif.), and all amino acid sequences of polypeptides
encoded by DNA molecules determined herein were predicted by
translation of a DNA sequence determined as above. Therefore, as is
known in the art for any DNA sequence determined by this automated
approach, any nucleotide sequence determined herein may contain
some errors. Nucleotide sequences determined by automation are
typically at least about 90% identical, more typically at least
about 95% to at least about 99.9% identical to the actual
nucleotide sequence of the sequenced DNA molecule. The actual
sequence can be more precisely determined by other approaches
including manual DNA sequencing methods well known in the art. As
is also known in the art, a single insertion or deletion in a
determined nucleotide sequence compared to the actual sequence will
cause a frame shift in translation of the nucleotide sequence such
that the predicted amino acid sequence encoded by a determined
nucleotide sequence will be completely different from the amino
acid sequence actually encoded by the sequenced DNA molecule,
beginning at the point of such an insertion or deletion.
By "nucleotide sequence" of a nucleic acid molecule or
polynucleotide is intended, for a DNA molecule or polynucleotide, a
sequence of deoxyribonucleotides, and for an RNA molecule or
polynucleotide, the corresponding sequence of ribonucleotides (A,
G, C and U), where each thymidine deoxyribonucleotide (T) in the
specified deoxyribonucleotide sequence is replaced by the
ribonucleotide uridine (U).
Using the information provided herein, such as the nucleotide
sequence in FIGS. 1A, 1B, and 1C (SEQ ID NO:1), a nucleic acid
molecule of the present invention encoding a follistatin-3
polypeptide may be obtained using standard cloning and screening
procedures, such as those for cloning cDNAs using mRNA as starting
material. Illustrative of the invention, the nucleic acid molecule
described in FIGS. 1A, 1B, and 1C (SEQ ID NO:1) was discovered in a
cDNA library derived from Hodgkin's Lymphoma.
Additional clones of the same gene were also identified in cDNA
libraries from the following cells and tissues: synovial
fibroblasts, gall bladder, resting and serum-induced smooth muscle,
testes, Merkel cells, HEL cells, hippocampus, TNF-.alpha.- and
IFN-induced epithelial cells, keratinocyte, amygdala depression,
HL-60 cells, hepatoma, progesterone-treated epidermal cells,
endothelial cells, HSC172 cells, epithelioid sarcoma, activated
T-cells, breast lymph node, pancreatic carcinoma, fetal dura mater,
fetal lung, epididymis, placenta, dendritic cells, rejected kidney,
and uterine cancer.
The determined nucleotide sequence of the follistatin-3 cDNA of
FIGS. 1A, 1B, and 1C (SEQ ID NO:1) contains an open reading frame
encoding a protein of 263 amino acid residues, with an initiation
codon at nucleotide positions 19-21 of the nucleotide sequence in
FIG. 1A (SEQ ID NO:1), and a deduced molecular weight of about 27.7
kDa. The amino acid sequence of the follistatin-3 protein shown in
SEQ ID NO:2 is about 43.2% identical to human mRNA for
follistatin-1 (FIG. 2; Shimasaki, S., et al., Proc. Natl. Acad.
Sci. U.S.A. 85:4218-4222 (1988); GenBank Accession No. J03771).
The open reading frame of the follistatin-3 gene shares sequence
homology with the translation product of the human mRNA for
follistatin-1 (FIG. 2; SEQ ID NO:3). The homology between
follistatin-1 and follistatin-3 indicates that follistatin-3 may
also be involved in a physiological regulation of cell growth and
differentiation, particularly with regard to cells of the
reproductive system.
As one of ordinary skill would appreciate, due to the possibilities
of sequencing errors discussed above, the actual complete
follistatin-3 polypeptide encoded by the deposited cDNA, which
comprises about 263 amino acids, may be somewhat longer or shorter.
More generally, the actual open reading frame may be anywhere in
the range of +20 amino acids, more likely in the range of +10 amino
acids, of that predicted from either the methionine codon from the
N-terminus shown in FIG. 1A (SEQ ID NO:1). It will further be
appreciated that, depending on the analytical criteria used for
identifying various functional domains, the exact "address" of the
mature form of the follistatin-3 polypeptide may differ slightly
from the predicted positions above. For example, the exact location
of the cleavage site of the precursor form of the mature
follistatin-3 molecule shown in SEQ ID NO:2 may vary slightly
(e.g., the address may "shift" by about 6 residues, depending on
the criteria used to define the cleavage site. In this case, the
ends of the signal peptide and the beginning of the mature
follistatin-3 molecule were predicted using the HGSI SignalP
computer algorithm. One of skill in the art will realize that
another widely accepted computer algorithm used to predict
potential sites of polypeptide cleavage, PSORT, will predict the
cleavage of an N-terminal signal peptide from the follistatin-3
polypeptide at a point slightly different from that predicted by
the HGSI SignalP algorithm. In either case, as discussed further
below, the invention further provides polypeptides having various
residues deleted from the N-terminus of the complete polypeptide,
including polypeptides corresponding to either of the predicted
mature follistatin-3 polypeptides described herein.
The amino acid sequence of the complete follistatin-3 protein
includes a leader sequence and a mature protein, as shown in SEQ ID
NO:2. Morein particular, the present invention provides nucleic
acid molecules encoding a mature form of the follistatin-3 protein.
Thus, according to the signal hypothesis, once export of the
growing protein chain across the rough endoplasmic reticulum has
been initiated, proteins secreted by mammalian cells have a signal
or secretory leader sequence which is cleaved from the complete
polypeptide to produce a secreted "mature" form of the protein.
Most mammalian cells and even insect cells cleave secreted proteins
with the same specificity. However, in some cases, cleavage of a
secreted protein is not entirely uniform, which results in two or
more mature species of the protein. Further, it has long been known
that the cleavage specificity of a secreted protein is ultimately
determined by the primary structure of the complete protein, that
is, it is inherent in the amino acid sequence of the polypeptide.
Therefore, the present invention provides a nucleotide sequence
encoding the mature follistatin-3 polypeptide having the amino acid
sequence encoded by the cDNA clone contained in the host identified
as ATCC.RTM. Deposit No. 209197. By the "mature follistatin-3
polypeptide having the amino acid sequence encoded by the cDNA
clone in ATCC.RTM. Deposit No. 209197" is meant the mature form(s)
of the follistatin-3 protein produced by expression in a mammalian
cell (e.g., COS cells, as described below) of the complete open
reading frame encoded by the human DNA sequence of the clone
contained in the vector in the deposited host.
In addition, methods for predicting whether a protein has a
secretory leader as well as the cleavage point for that leader
sequence are available. For instance, the method of McGeoch (Virus
Res. 3:271-286 (1985)) uses the information from a short N-terminal
charged region and a subsequent uncharged region of the complete
(uncleaved) protein. The method of von Heinje (Nucleic Acids Res.
14:4683-4690 (1986)) uses the information from the residues
surrounding the cleavage site, typically residues -13 to +2 where
+1 indicates the amino terminus of the mature protein. The accuracy
of predicting the cleavage points of known mammalian secretory
proteins for each of these methods is in the range of 75-80% (von
Heinje, supra). However, the two methods do not always produce the
same predicted cleavage point(s) for a given protein.
In the present case, the deduced amino acid sequence of the
complete follistatin-3 polypeptide was analyzed by the HGSI SignalP
algorithm, which is an expert system for predicting the cellular
location of a protein based on the amino acid sequence. As part of
this computational prediction of localization, the methods of
McGeoch and von Heinje are incorporated. Thus, the computation
analysis above predicted a single cleavage site within the complete
amino acid sequence shown in SEQ ID NO:2 (see above
discussion).
As indicated, nucleic acid molecules of the present invention may
be in the form of RNA, such as mRNA, or in the form of DNA,
including, for instance, cDNA and genomic DNA obtained by cloning
or produced synthetically. The DNA may be double-stranded or
single-stranded. Single-stranded DNA or RNA may be the coding
strand, also known as the sense strand, or it may be the non-coding
strand, also referred to as the anti-sense strand.
By "isolated" nucleic acid molecule(s) is intended a nucleic acid
molecule, DNA or RNA, which has been removed from its native
environment For example, recombinant DNA molecules contained in a
vector are considered isolated for the purposes of the present
invention. Further examples of isolated DNA molecules include
recombinant DNA molecules maintained in heterologous host cells or
purified (partially or substantially) DNA molecules in solution.
Isolated RNA molecules include in vivo or in vitro RNA transcripts
of the DNA molecules of the present invention. Isolated nucleic
acid molecules according to the present invention further include
such molecules produced synthetically.
Isolated nucleic acid molecules of the present invention include
DNA molecules comprising an open reading frame (ORF) with an
initiation codon at positions 19-21 of the nucleotide sequence
shown in FIG. 1A (SEQ ID NO:1).
Also included are DNA molecules comprising the coding sequence for
the predicted mature follistatin-3 protein shown at positions 1-237
of SEQ ID NO:2.
In addition, isolated nucleic acid molecules of the invention
include DNA molecules which comprise a sequence substantially
different from those described above but which, due to the
degeneracy of the genetic code, still encode the follistatin-3
protein. Of course, the genetic code and species-specific codon
preferences are well known in the art. Thus, it would be routine
for one skilled in the art to generate the degenerate variants
described above, for instance, to optimize codon expression for a
particular host (e.g., change codons in the human mRNA to those
preferred by a bacterial host such as E. coli).
In another aspect, the invention provides isolated nucleic acid
molecules encoding the follistatin-3 polypeptide having an amino
acid sequence encoded by the cDNA clone contained in the plasmid
deposited as ATCC.RTM. Deposit No. 209197 on Aug. 8, 1997.
Preferably, this nucleic acid molecule will encode the mature
polypeptide encoded by the above-described deposited cDNA
clone.
The invention further provides an isolated nucleic acid molecule
having the nucleotide sequence shown in FIGS. 1A, 1B, and 1C (SEQ
ID NO:1) or the nucleotide sequence of the follistatin-3 cDNA
contained in the above-described deposited clone, or a nucleic acid
molecule having a sequence complementary to one of the above
sequences. Such isolated molecules, particularly DNA molecules, are
useful as probes for gene mapping, by in situ hybridization with
chromosomes, and for detecting expression of the follistatin-3 gene
in human tissue, for instance, by Northern blot analysis.
The present invention is further directed to nucleic acid molecules
encoding portions of the nucleotide sequences described herein as
well as to fragments of the isolated nucleic acid molecules
described herein. In particular, the invention provides a
polynucleotide having a nucleotide sequence representing the
portion of SEQ ID NO: 1 which consists of positions 1-810 of SEQ ID
NO:1.
In addition, the invention provides nucleic acid molecules having
nucleotide sequences related to extensive portions of SEQ ID NO:1
which have been determined from the following related cDNA clones:
HHPDX66R (SEQ ID NO:4), HDTAH61R (SEQ ID NO:5), HSBAV55R (SEQ ID
NO:6), HUKFS32R (SEQ ID NO:7), HOOAD78R (SEQ ID NO:8), HAQAG52R
(SEQ ID NO:9), HTLEJ56R (SEQ ID NO:10), HLMNX90R (SEQ ID
NO:11).
Further, the invention includes a polynucleotide comprising any
portion of at least about 30 nucleotides, preferably at least about
50 nucleotides, of SEQ ID NO:1 from. residue 1 to 500. More
preferably, the invention includes a polynucleotide comprising
nucleotide residues 100-500, 200-500, 300-500, 400-500, 100-400,
200-400, 300-400, 100-300, 200-300, 100-200, 100-2495, 250-2495,
500-2495, 1000-2495, 1500-2495, 2000-2495, 100-2000, 250-2000,
500-2000, 1000-2000, 1500-2000, 100-1500, 250-1500, 500-1500,
1000-1500, 100-1000, 250-1000, and 500-1000.
More generally, by a fragment of an isolated nucleic acid molecule
having the nucleotide sequence of the deposited cDNA or the
nucleotide sequence shown in FIGS. 1A, 1B, and 1C (SEQ ID NO:1) is
intended fragments at least about 15 nt, and more preferably at
least about 20 nt, still more preferably at least about 30 nt, and
even more preferably, at least about 40 nt in length which have
uses that include, but are not limited to, as diagnostic probes and
primers as discussed herein. Of course, larger fragments 50-300 nt
in length are also useful according to the present invention as are
fragments. corresponding to most, if not all of the nucleotide
sequence of the deposited cDNA or as shown in FIGS. 1A, 1B, and 1C
(SEQ ID NO:1). By a fragment at least 20 nt in length, for example,
is intended fragments which include 20 or more contiguous bases
from the nucleotide sequence of the deposited cDNA or the
nucleotide sequence as shown in FIGS. 1A, 1B, and 1C (SEQ ID NO:1).
Preferred nucleic acid fragments of the present invention include
nucleic acid molecules encoding epitope-bearing portions of the
follistatin-3 polypeptide as identified in FIG. 3 and described in
more detail below.
In specific embodiments, the polynucleotide fragments of the
invention encode a polypeptide which demonstrates a follistatin-3
functional activity. By a polypeptide demonstrating follistatin-3
"functional activity" is meant, a polypeptide capable of displaying
one or more known functional activities associated with a complete,
mature or active form of the follistatin-3 polypeptide. Such
functional activities include, but are not limited to, biological
activity ((e.g., modulating the follicle stimulating hormone (FSH)
synthetic pathway, increasing estradiol production, binding
activin, stimulating of gonadotropin biosynthesis and secretion,
regulating ovarian and placental steroidogenesis, and oocyte and
spermatogonial maturation factor)), antigenicity [ability to bind
(or compete with a follistatin-3 polypeptide for binding) to an
anti-follistatin-3 antibody], immuunogenicity (ability to generate
antibody which binds to a follistatin-3 polypeptide), the ability
to form polymers with other follistatin-3 or inhibin or TGF-.beta.
polypeptides, and ability to bind to a receptor or ligand (e.g., an
inhibin) for a follistatin-3 polypeptide.
Preferred nucleic acid fragments of the present invention also
include nucleic acid molecules encoding one or more of the
following domains of follistatin-3: amino acid residues 7-16,
34-45, 78-86, 91-100, 108-122, 131-145, 156-169, 184-192, and
196-210 of SEQ ID NO:2.
In specific embodiments, the polynucleotide fragments of the
invention encode antigenic regions. Non-limiting examples of
antigenic polypeptides or peptides that can be used to generate
follistatin-3-specific antibodies include: a polypeptide comprising
amino acid residues: Leu-14 to Ala-20, Ser-46 to Ile-55, Gly-88 to
Pro-97, Gly-l 13 to Leu-133, Arg-138 to Glu-146, Pro-177 to
Thr-191, and Gly-219 to Val-237 of SEQ ID NO:2.
In additional embodiments, the polynucleotides of the invention
encode functional attributes of follistatin-3. Preferred
embodiments of the invention in this regard include fragments that
comprise alpha-helix and alpha-helix forming regions
("alpha-regions"), beta-sheet and beta-sheet forming regions
("beta-regions"), turn and turn-forming regions ("turn-regions"),
coil and coil-forming regions ("coil-regions"), hydrophilic
regions, hydrophobic regions, alpha amphipathic regions, beta
amphipathic regions, flexible regions, surface-forming regions and
high antigenic index regions of follistatin-3.
The data representing the structural or functional attributes of
follistatin-3 set forth in FIG. 3 and/or Table I, as described
above, was generated using the various modules and algorithms of
the DNA*STAR set on default parameters. In a preferred embodiment,
the data presented in columns VIII, IX, XIII, and XIV of Table I
can be used to determine regions of follistatin-3 which exhibit a
high degree of potential for antigenicity. Regions of high
antigenicity are determined from the data presented in columns
VIII, IX, XIII, and/or IV by choosing values which represent
regions of the polypeptide which are likely to be exposed on the
surface of the polypeptide in an environment in which antigen
recognition may occur in the process of initiation of an immune
response.
Certain preferred regions in these regards are set out in FIG. 3,
but may, as shown in Table I, be represented or identified by using
tabular representations of the data presented in FIG. 3. The
DNA*STAR computer algorithm used to generate FIG. 3 (set on the
original default parameters) was used to present the data in FIG. 3
in a tabular format (See Table I). The tabular format of the data
in FIG. 3 may be used to easily determine specific boundaries of a
preferred region.
The above-mentioned preferred regions set out in FIG. 3 and in
Table I include, but are not limited to, regions of the
aforementioned types identified by analysis of the amino acid
sequence set out in FIGS. 1A, 1B, and 1C. As set out in FIG. 3 and
in Table I, such preferred regions include Garnier-Robson
alpha-regions, beta-regions, turn-regions, and coil-regions,
Chou-Fasman alpha-regions, beta-regions, and coil-regions,
Kyte-Doolittle hydrophilic regions and hydrophobic regions,
Eisenberg alpha- and beta-amphipathic regions, Karplus-Schulz
flexible regions, Emini surface-forming regions and Jameson-Wolf
regions of high antigenic index.
Among highly preferred fragments in this regard are those that
comprise reigons of follistatin-3 that combine several structural
features, such as two, three, four, five or more of the features
set out above and in Table I.
TABLE I Res Position I II III IV V VI VII VIII IX X XI XII XIII XIV
Met 1 . . B . . . . 0.31 -0.24 * * . 1.07 1.11 Arg 2 . . B . . . .
0.49 -0.17 * * . 1.13 0.88 Pro 3 . . . . T . . 0.53 -0.17 * * .
1.89 1.06 Gly 4 . . . . T . . 0.71 -0.17 * * . 2.10 1.06 Ala 5 . .
. . . T C 0.29 -0.36 . * F 1.89 0.84 Pro 6 . . . . . T C 0.60 0.33
. * F 1.08 0.45 Gly 7 . . . . . T C 0.28 0.81 . * F 0.57 0.48 Pro 8
. . B . . T . -0.32 0.81 . . F 0.16 0.73 Leu 9 . . B . . . . -0.19
1.00 . . F -0.25 0.39 Trp 10 . . B . . . . 0.11 1.00 . . . -0.40
0.61 Pro 11 . . B . . . . -0.02 1.49 . . . -0.40 0.41 Leu 12 . . B
. . T . -0.27 1.49 . . . -0.20 0.49 Pro 13 . . . . T T . -0.87 1.30
. . . 0.20 0.48 Trp 14 . . . . T T . -0.64 1.07 . . . 0.20 0.25 Gly
15 . . . . . T C -0.64 1.14 . . . 0.00 0.31 Ala 16 . A . . . . C
-1.02 1.37 . . . -0.40 0.21 Leu 17 . A B . . . . -1.07 1.44 . . .
-0.60 0.20 Ala 18 . A B B . . . -1.20 1.17 . . . -0.60 0.15 Trp 19
. A B B . . . -1.61 1.17 . . . -0.60 0.15 Ala 20 . A B B . . .
-2.12 1.46 . . . -0.60 0.16 Val 21 . A B B . . . -1.83 1.41 . . .
-0.60 0.11 Gly 22 . A B B . . . -1.32 1.30 . . . -0.60 0.15 Phe 23
. . B B . . . -1.33 0.77 . . . -0.60 0.19 Val 24 . . B B . . .
-1.39 0.89 . . . -0.60 0.26 Ser 25 . . B . . . . -1.10 0.67 * . .
-0.40 0.26 Ser 26 . . B . . . . -0.59 0.63 . . F -0.25 0.40 Met 27
. . . . T . . -0.24 0.27 . . F 0.45 0.53 Gly 28 . . . . T T . 0.24
0.03 . . F 0.82 0.64 Ser 29 . . . . T T . 0.51 0.07 . . F 0.99 0.74
Gly 30 . . . . . T C 0.60 0.19 . . F 0.96 0.76 Asn 31 . . . . . T C
0.56 -0.00 . . F 1.88 1.18 Pro 32 . . . . . . C 0.81 -0.00 . . F
1.70 0.87 Ala 33 . . . . . T C 0.30 0.04 . . F 1.13 0.87 Pro 34 . .
. . T T . -0.07 0.26 . . F 1.16 0.40 Gly 35 . . . . T T . -0.01
0.43 * . F 0.69 0.14 Gly 36 . . B . . T . -0.82 0.91 * . F 0.12
0.15 Val 37 . A B . . . . -0.61 1.10 * . . -0.60 0.08 Cys 38 . A B
. . . . -0.02 1.07 * . . -0.60 0.14 Trp 39 . A B . . . . -0.16 1.04
* . . -0.60 0.24 Leu 40 . A B . . . . 0.19 1.04 * . . -0.32 0.32
Gln 41 . . B . . T . 0.53 0.80 * . F 0.66 1.02 Gln 42 . . . . T T .
0.80 0.23 * . F 1.64 1.68 Gly 43 . . . . T T . 1.16 -0.19 * . F
2.52 2.06 Gln 44 . . . . T T . 0.78 -0.39 * . F 2.80 1.72 Glu 45 .
. . . T . . 1.29 -0.21 * . F 2.17 0.53 Ala 46 . . . . T T . 0.48
-0.23 * . F 2.09 0.72 Thr 47 . . B . . T . -0.38 0.03 . . . 0.66
0.34 Cys 48 . . B . . T . -0.84 0.27 . . . 0.38 0.15 Ser 49 . . B .
. T . -0.84 0.96 . . . -0.20 0.12 Leu 50 . . B B . . . -1.16 0.86 .
* . -0.60 0.14 Val 51 . . B B . . . -0.57 0.86 . * . -0.60 0.39 Leu
52 . . B B . . . -1.11 0.29 . * . -0.30 0.48 Gln 53 . . B B . . .
-0.76 0.54 * * F -0.45 0.43 Thr 54 . . B B . . . -0.34 0.34 * . F
-0.15 0.85 Asp 55 . . B B . . . -0.12 -0.30 * * F 0.60 2.01 Val 56
. A B B . . . 0.73 -0.49 * . F 0.60 1.17 Thr 57 . A B B . . . 0.88
-0.89 * * F 0.90 1.41 Arg 58 . A B B . . . 0.21 -0.80 * * F 0.75
0.45 Ala 59 . A B B . . . -0.07 -0.23 * * . 0.30 0.33 Glu 60 . A B
B . . . -0.37 -0.37 * * . 0.30 0.23 Cys 61 . A B . . . . 0.14 -0.47
* * . 0.55 0.16 Cys 62 . . . . T T . 0.46 -0.04 * * . 1.60 0.15 Ala
63 . . . . T T . -0.54 -0.14 * * . 1.85 0.14 Ser 64 . . . . T T .
0.04 0.54 . * F 1.35 0.19 Gly 65 . . . . T T . -0.27 -0.03 . * F
2.50 0.58 Asn 66 . . . . T T . -0.19 -0.11 . * F 2.25 0.83 Ile 67 .
. B . . T . 0.19 -0.11 * * F 1.60 0.62 Asp 68 . . B . . T . 0.48
0.41 * * F 0.45 0.66 Thr 69 . . B . . T . 0.78 0.37 * * F 0.50 0.55
Ala 70 . . B . . . . 0.31 0.37 * * . 0.05 1.26 Trp 71 . . B . . T .
-0.00 0.37 * . . 0.10 0.62 Ser 72 . . B . . T . 0.86 0.86 * . .
0.20 0.62 Asn 73 . . B . . T . 0.64 0.87 * . . 0.20 0.84 Leu 74 . .
. . . T C 0.61 0.80 * . . 0.43 1.24 Thr 75 . . . . . . C 1.20 0.31
* . . 0.66 0.91 His 76 . . . . . T C 1.53 0.33 * . F 1.29 0.91 Pro
77 . . . . . T C 0.94 -0.07 * . F 2.32 2.22 Gly 78 . . . . T T .
0.94 -0.07 * * F 2.80 1.08 Asn 79 . . . . T T . 0.94 -0.16 * . F
2.52 1.27 Lys 80 . . B . . . . 0.44 0.03 * * F 0.89 0.68 Ile 81 . .
B . . . . 0.13 0.29 . . F 0.61 0.57 Asn 82 . . B . . . . -0.36 0.29
. * . 0.38 0.35 Leu 83 . . B B . . . -0.82 0.67 . . . -0.60 0.15
Leu 84 . . B B . . . -1.17 1.36 . * . -0.60 0.18 Gly 85 . . B B . .
. -2.02 1.10 . * . -0.60 0.11 Phe 86 . . B B . . . -1.99 1.39 . . .
-0.60 0.11 Leu 87 . . B B . . . -2.02 1.34 . . . -0.60 0.10 Gly 88
. . B B . . . -1.88 1.16 * . . -0.60 0.13 Leu 89 . . B B . . .
-1.88 1.30 . . . -0.60 0.08 Val 90 . . B B . . . -1.74 1.20 * . .
-0.60 0.08 His 91 . . B B . . . -1.71 0.94 * . . -0.60 0.13 Cys 92
. . B B . . . -0.86 1.09 . . . -0.60 0.08 Leu 93 . . B B . . .
-0.51 0.40 . . . 0.01 0.23 Pro 94 . . . B T . . -0.00 -0.24 . . .
1.32 0.28 Cys 95 . . . . T T . 0.19 -0.36 . . . 2.03 0.70 Lys 96 .
. . . T T . 0.22 -0.36 . . F 2.49 0.46 Asp 97 . . . . T T . 0.54
-1.04 * . F 3.10 0.49 Ser 98 . . . . T T . 0.50 -1.04 * . F 2.79
0.91 Cys 99 . . . . T T . 0.71 -0.97 * . F 2.48 0.34 Asp 100 . . B
. . T . 0.71 -0.97 * . F 1.77 0.35 Gly 101 . . B . . T . 0.32 -0.40
* . F 1.47 0.14 Val 102 . . B . . T . 0.11 -0.36 * . . 1.32 0.26
Glu 103 . . B . . . . 0.07 -0.50 * . . 1.73 0.24 Cys 104 . . . . T
. . 0.78 -0.07 * . F 2.29 0.24 Gly 105 . . . . T T . 0.19 -0.50 * .
F 3.10 0.64 Pro 106 . . . . T T . -0.13 -0.64 * . F 2.79 0.38 Gly
107 . . . . T T . 0.83 -0.07 * . F 2.18 0.38 Lys 108 . . . . T T .
0.23 -0.64 * . F 2.17 0.74 Ala 109 . A B . . . . 0.09 -0.46 * . .
0.61 0.48 Cys 110 . A B . . . . 0.09 -0.20 * . . 0.30 0.40 Arg 111
. A B . . . . -0.04 -0.20 * . . 0.30 0.20 Met 112 . A B . . . .
0.41 0.23 * . . -0.30 0.19 Leu 113 . A . . T . . 0.16 -0.27 * * .
1.04 0.70 Gly 114 . A . . T . . 0.86 -0.41 * * F 1.53 0.55 Gly 115
. . . . T . . 0.86 -0.41 * * F 2.22 1.10 Arg 116 . . . . . T C 0.74
-0.46 * * F 2.41 0.71 Pro 117 . . . . T T . 0.68 -1.14 . * F 3.40
1.25 Arg 118 . . . . T T . 0.90 -1.00 . * F 2.91 0.68 Cys 119 . . B
. . T . 1.03 -0.93 . * . 2.02 0.35 Glu 120 . . B . . . . 1.38 -0.50
. * . 1.73 0.35 Cys 121 . . B . . . . 0.60 -0.93 . * . 1.64 0.30
Ala 122 . . B . . T . 0.51 -0.36 . * . 1.45 0.30 Pro 123 . . . . T
T . 0.06 -0.54 . * F 2.55 0.23 Asp 124 . . . . T T . -0.09 -0.11 .
. F 2.50 0.43
Cys 125 . . . . T T . -0.30 -0.00 . . F 2.25 0.35 Ser 126 . . . . T
. . -0.22 -0.07 * * F 1.80 0.35 Gly 127 . . . . T . . 0.48 -0.00 *
* F 1.55 0.21 Leu 128 . . B . . . . -0.12 -0.00 * * . 0.75 0.77 Pro
129 . . B . . . . -0.12 0.11 . * . -0.10 0.47 Ala 130 . . B . . . .
-0.31 0.13 . * . -0.10 0.83 Arg 131 . . B B . . . -0.68 0.34 . * .
-0.30 0.74 Leu 132 . . B B . . . -0.68 0.23 . * . -0.30 0.26 Gln
133 . . B B . . . -0.17 0.23 * * . -0.30 0.25 Val 134 . . B B . . .
0.04 0.11 * * . -0.30 0.17 Cys 135 . . B B . . . 0.29 0.11 * * .
-0.02 0.35 Gly 136 . . B . . T . -0.41 -0.14 * * F 1.41 0.20 Ser
137 . . . . T T . 0.09 -0.04 . . F 2.09 0.27 Asp 138 . . . . T T .
-0.16 -0.20 . * F 2.37 0.73 Gly 139 . . . . T T . 0.81 -0.01 . . F
2.80 1.16 Ala 140 . . . . T . . 1.48 -0.44 . . F 2.32 1.70 Thr 141
. . B . . . . 1.82 -0.83 . . . 1.99 1.70 Tyr 142 . . B . . T . 1.46
-0.83 . * . 2.11 2.97 Arg 143 . . B . . T . 1.46 -0.69 . * F 2.18
1.57 Asp 144 . . B . . T . 0.99 -1.19 . * F 2.10 1.89 Glu 145 . . B
. . T . 1.69 -0.99 . * . 2.00 0.99 Cys 146 . A B . . . . 1.41 -1.74
. * . 1.40 0.99 Glu 147 A A . . . . . 1.07 -1.24 . * . 1.20 0.60
Leu 148 A A . . . . . 1.07 -0.74 . * . 1.00 0.35 Arg 149 A A . . .
. . 0.40 -0.74 . * . 0.95 1.28 Ala 150 A A . . . . . 0.51 -0.74 * *
. 0.60 0.40 Ala 151 . A . . T . . 0.83 -0.74 . * . 1.00 0.94 Arg
152 . A . . T . . 0.80 -1.00 . * . 1.00 0.48 Cys 153 . A . . T . .
1.40 -0.50 . * . 1.27 0.64 Arg 154 . A . . T . . 1.29 -0.57 . * .
1.54 0.98 Gly 155 . A . . T . . 1.07 -1.07 . * F 1.96 0.84 His 156
. . . . . T C 1.36 -0.39 . * F 2.28 1.29 Pro 157 . . . . . T C 0.39
-0.57 . * F 2.70 0.88 Asp 158 . . . . T T . 0.46 0.07 * * F 1.73
0.66 Leu 159 . . B . . T . 0.10 0.26 * * . 0.91 0.48 Ser 160 . . B
B . . . 0.56 0.51 * * . -0.06 0.49 Val 161 . . B B . . . 0.24 0.09
. * . -0.03 0.57 Met 162 . . B B . . . 0.57 0.51 . * . -0.26 0.69
Tyr 163 . . B . . T . -0.10 -0.17 * * . 1.53 1.00 Arg 164 . . B . .
T . 0.82 0.01 . * . 1.12 0.72 Gly 165 . . . . T T . 1.17 -0.63 . *
F 3.06 1.43 Arg 166 . . . . T T . 1.72 -1.24 . * F 3.40 1.83 Cys
167 . . . . T . . 1.66 -1.61 * * F 2.86 1.25 Arg 168 . . . . T T .
1.90 -1.04 * * F 2.57 0.68 Lys 169 . . . . T T . 1.76 -1.47 * * F
2.23 0.60 Ser 170 . . . . T T . 1.24 -0.97 * * F 2.04 1.52 Cys 171
. . . . T T . 0.28 -0.90 * * . 1.40 0.58 Glu 172 . . B B . . . 0.28
-0.26 * . . 0.30 0.21 His 173 . . B B . . . -0.04 0.31 . . . -0.30
0.09 Val 174 . . B B . . . 0.02 0.36 . * . -0.02 0.25 Val 175 . . B
B . . . 0.11 -0.21 . * . 0.86 0.28 Cys 176 . . B . . T . 0.78 0.21
. * . 0.94 0.32 Pro 177 . . . . T T . 0.48 0.11 . * F 1.77 0.74 Arg
178 . . . . T T . -0.16 -0.14 . * F 2.80 1.34 Pro 179 . . . . T T .
-0.16 -0.21 . * F 2.52 1.34 Gln 180 . . . B T . . -0.16 -0.14 * * F
1.69 0.64 Ser 181 . . B B . . . 0.51 0.07 * * F 0.41 0.24 Cys 182 .
. B B . . . 0.72 0.07 * * . -0.02 0.26 Val 183 . . B B . . . 0.30
0.04 * * . -0.30 0.26 Val 184 . . B B . . . 0.17 0.13 . . . 0.02
0.28 Asp 185 . . B B . . . -0.13 0.17 . . F 0.41 0.52 Gln 186 . . B
. . T . -0.42 -0.01 . . F 1.69 0.94 Thr 187 . . . . T T . 0.21
-0.16 . . F 2.52 1.28 Gly 188 . . . . T T . 0.40 -0.30 . . F 2.80
1.04 Ser 189 . . . . T T . 0.40 0.27 . . F 1.77 0.32 Ala 190 . . B
B T . . -0.46 0.51 . . . 0.64 0.17 His 191 . . B B . . . -1.12 0.67
* . . -0.04 0.12 Cys 192 . . B B . . . -0.70 0.81 * * . -0.32 0.05
Val 193 . . B B . . . -0.94 0.43 * . . -0.60 0.10 Val 194 . . B B .
. . -1.23 0.43 * . . -0.60 0.07 Cys 195 . . B B . . . -0.86 0.43 *
. . -0.60 0.14 Arg 196 . . B B . . . -1.49 0.29 * . . -0.30 0.28
Ala 197 . . B B . . . -1.03 0.21 * . . -0.30 0.20 Ala 198 . . B . .
T . -1.03 -0.00 * . . 0.70 0.59 Pro 199 . . B . . T . -0.39 0.07 *
. . 0.10 0.22 Cys 200 . . B . . T . -0.02 0.50 * . . -0.20 0.34 Pro
201 . . B . . T . -0.43 0.39 * . . 0.10 0.45 Val 202 . . B . . . .
-0.06 0.27 . . F 0.05 0.39 Pro 203 . . . . T . . 0.19 0.27 . . F
0.88 1.13 Ser 204 . . . . T . . 0.40 0.13 . . F 1.01 0.72 Ser 205 .
. . . . T C 1.07 0.10 * . F 1.44 1.69 Pro 206 . . . . T T . 0.47
-0.54 * . F 2.82 1.89 Gly 207 . . . . T P . 0.66 -0.29 . . F 2.80
1.16 Gln 208 . . B . . T . 0.52 -0.10 . . F 1.97 0.47 Glu 209 . . B
. . . . 0.82 -0.06 . . F 1.49 0.30 Leu 210 . . B . . . . 1.12 -0.09
. . F 1.37 0.48 Cys 211 . . B . . T . 1.33 -0.11 . . F 1.45 0.45
Gly 212 . . . . T T . 0.82 -0.11 . . F 1.73 0.42 Asn 213 . . . . T
T . 0.51 0.53 . * F 0.99 0.38 Asn 214 . . . . T T . 0.27 0.33 . * F
1.60 1.01 Asn 215 . . . B T . . 0.19 0.51 . . F 0.74 1.60 Val 216 .
. B B . . . 0.56 0.77 * . . -0.12 0.70 Thr 217 . . B B . . . 0.60
0.76 * . . -0.28 0.58 Tyr 218 . . B B . . . -0.07 0.74 . . . -0.44
0.48 Ile 219 . . B B . . . -0.10 0.91 . . . -0.60 0.35 Ser 220 . .
B . . T . -0.70 0.77 * * . -0.20 0.33 Ser 221 . . B . . T . 0.27
0.90 * . . -0.20 0.21 Cys 222 . . B . . T . 0.58 0.14 * . . 0.10
0.58 His 223 . . B . . T . 0.23 -0.14 * . . 0.70 0.75 Met 224 . . .
. T . . 0.81 -0.03 * . . 0.90 0.57 Arg 225 . . B B . . . 0.44 0.07
* * . -0.15 1.53 Gln 226 . . B B . . . 0.04 0.07 * . . -0.30 0.60
Ala 227 . . B B . . . -0.10 0.36 * . . -0.30 0.53 Thr 228 . . B B .
. . -0.41 0.43 * * . -0.60 0.22 Cys 229 . . B B . . . 0.30 0.86 * *
. -0.60 0.13 Phe 230 . . B B . . . -0.11 0.46 * . . -0.60 0.25 Leu
231 . . B B . . . -1.00 0.34 * . . -0.30 0.23 Gly 232 . . . . T T .
-0.76 0.54 * . . 0.20 0.30 Arg 233 . . . . T T . -1.30 0.40 . * F
0.65 0.34 Ser 234 . . . . T T . -0.52 0.26 . . F 0.65 0.31 Ile 235
. . B . . T . 0.14 -0.43 . . . 0.70 0.61 Gly 236 . . B B . . . 0.37
-0.36 . . . 0.30 0.42 Val 237 . . B B . . . 0.37 0.14 . . . -0.30
0.32 Arg 238 . . B B . . . -0.04 0.19 . * . -0.30 0.45 His 239 . .
B . . T . -0.41 -0.11 * * . 0.70 0.61 Ala 240 . . . . T T . -0.11
0.03 * * . 0.50 0.44 Gly 241 . . . . T T . -0.11 -0.11 * . . 1.10
0.23 Ser 242 . . . . T T . 0.43 0.31 * * . 0.80 0.16 Cys 243 . . .
. T T . 0.11 0.30 * . . 1.10 0.24 Ala 244 . . . . T T . 0.14 0.23 .
. . 1.40 0.37 Gly 245 . . . . . T C 0.73 -0.20 . . F 2.25 0.47 Thr
246 . . . . . T C 0.87 -0.59 . . F 3.00 1.53 Pro 247 . . . . . . C
0.96 -0.73 . . F 2.50 2.35 Glu 248 . . . . . . C 1.28 -0.80 . . F
2.50 3.67 Glu 249 . . . . . . C 1.52 -0.80 . . F 2.50 2.52 Pro 250
. . . . . T C 1.87 -0.86 . . F 2.70
1.61 Pro 251 . . . . . T C 1.88 -1.29 . . F 2.70 1.61 Gly 252 . . .
. . T C 1.50 -0.90 . . F 3.00 1.25 Gly 253 . . . . . T C 1.50 -0.40
. . F 2.25 0.81 Glu 254 . A . . . . C 1.50 -0.83 . . F 1.85 0.91
Ser 255 . A . . . . C 1.71 -1.26 . . F 1.70 1.60 Ala 256 A A . . .
. . 1.92 -1.69 * . F 1.20 2.79 Glu 257 A A . . . . . 2.27 -2.11 * .
F 0.90 2.79 Glu 258 A A . . . . . 1.91 -1.71 * . F 0.90 3.35 Glu
259 A A . . . . . 1.06 -1.31 * . F 0.90 2.87 Glu 260 A A . . . . .
0.97 -1.17 * . F 0.90 1.23 Asn 261 A A . . . . . 1.17 -0.74 * . .
0.60 0.91 Phe 262 A A . . . . . 0.78 -0.31 . . . 0.30 0.67 Val 263
A A . . . . . 0.39 0.11 . . . -0.30 0.49
In another aspect, the invention provides an isolated nucleic acid
molecule comprising a polynucleotide which hybridizes under
strigent hybridization condition to a portion of the polynucleotide
in a nucleic acid molecule of the invention described above, for
instance, the cDNA clone contained in ATCC.RTM. Deposit No. 209197,
a polynucleotide sequence encoding the follistatin-3 polypeptide
having the amino acid sequence depicted in FIGS. 1A-C (SEQ ID
NO:2), or fragments (i.e., portions) thereof (as described herein).
By "stringent hybridization conditions" is intended one-night
incubation an 42.degree. C. in a solution comprising: 50%
formamide. 5.times.SSC (750mM NaCl, 75 mM trisodium citrate), 50 mM
sodium phosphate (p7.6), 5.times. Denhardts solution, 10% dexuan
sulfate, and 20 .mu.g/ml denatured, Sheared salmon sperm DNA,
followed by washing the tilters in 0.1.times.SSC at about
65.degree. C.
By a polynucleotide which hybridizes to a "portion" of a
polynucleotide is intended a polynucleotide (either DNA or RNA)
hybridizing to at least about 15 nucleotides (nt), and more
preferably at least about 20 nt, still more preferably at least
about 30 nt, and even more preferably about 30-70 (e.g., 50) nt of
the reference polynucleotide. These are useful as diagnostic probes
and primers as discussed above and in more detail below.
By a portion of a polynucleotide of "at least 20 nt in length," for
example, is intended 20 or more contiguous nucleotides from the
nucleotide sequence of the reference polynucleotide (e.g., the
deposited cDNA or the nucleotide sequence as shown in FIGS. 1A, 1B,
and 1C (SEQ ID NO:1)). Of course, a polynucleotide which hybridizes
only to a poly A sequence (such as the 3' terminal poly(A) tract of
the follistatin-3 cDNA shown in FIGS. 1A, 1B, and 1C (SEQ ID
NO:1)), or to a complementary stretch of T (or U) residues, would
not be included in a polynucleotide of the invention used to
hybridize to a portion of a nucleic acid of the invention, since
such a polynucleotide would hybridize to any nucleic acid molecule
containing a poly (A) stretch or the complement thereof (e.g.,
practically any double-stranded cDNA clone).
In preferred embodiments, polynucleotides which hybridize to the
reference polynucleotides disclosed herein encode polypeptides
which either retain substantially the same functional or biological
activity as the mature form of the follistatin-3 polypeptide
encoded by the polynucleotide sequence depicted in FIGS. 1A, 1B,
and 1C (SEQ ID NO:1) or the clone contained in the deposit
(HDTAH85).
Alternative embodiments are directed to polynucleotides which
hybridize to the reference polynucleotide (i.e., a polynucleotide
sequence disclosed herein), but do not retain biological activity.
While these polynucleotides do not retain biological activity, they
have uses, such as, for example, as probes for the polynucleotides
of SEQ ID NO:1, for recovery of the polynucleotides, as diagnostic
probes, and as PCR primers.
As indicated, nucleic acid molecules of the present invention which
encode a follistatin-3 polypeptide may include, but are not limited
to, those encoding the amino acid sequence of the mature
polypeptide, by itself; and the coding sequence for the mature
polypeptide and additional sequences, such as those encoding the
about 26 amino acid leader or secretory sequence, such as a pre-,
or pro- or prepro-protein sequence; the coding sequence of the
mature polypeptide, with or without the aforementioned additional
coding sequences.
Also encoded by nucleic acids of the invention are the above
protein sequences together with additional, non-coding sequences,
including for example, but not limited to introns and non-coding 5'
and 3' sequences, such as the transcribed, non-translated sequences
that play a role in transcription, mRNA processing, including
splicing and polyadenylation signals, for example--ribosome binding
and stability of mRNA; an additional coding sequence which codes
for additional amino acids, such as those which provide additional
functionalities.
Thus, the sequence encoding the polypeptide may be fused to a
marker sequence, such as a sequence encoding a peptide which
facilitates purification of the fused polypeptide. In certain
preferred embodiments of this aspect of the invention, the marker
amino acid sequence is a hexa-histidine peptide, such as the tag
provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue,
Chatsworth, Calif., 91311), among others, many of which are
commercially available. As described by Gentz and colleagues (Proc.
Natl. Acad. Sci. USA 86:821-824 (1989)), for instance,
hexa-histidine provides for convenient purification of the fusion
protein. The "HA" tag is another peptide useful for purification
which corresponds to an epitope derived from the influenza
hemagglutinin protein, which has been described by Wilson and
coworkers (Cell 37:767 (1984)). As discussed below, other such
fusion proteins include the follistatin-3 fused to Fc at the N- or
C-terminus.
The present invention further relates to variants of the nucleic
acid molecules of the present invention, which encode portions,
analogs or derivatives of the follistatin-3 polypeptide. Variants
may occur naturally, such as a natural allelic variant. By an
"allelic variant" is intended one of several alternate forms of a
gene occupying a given locus on a chromosome of an organism (Genes
II, Lewin, B., ed., John Wiley & Sons, New York (1985)).
Non-naturally occurring variants may be produced using art-known
mutagenesis techniques.
Such variants include those produced by nucleotide substitutions,
deletions or additions. The substitutions, deletions or additions
may involve one or more nucleotides. The variants may be altered in
coding regions, non-coding regions, or both. Alterations in the
coding regions may produce conservative or non-conservative amino
acid substitutions, deletions or additions. Especially preferred
among these are silent substitutions, additions and deletions,
which do not alter the properties and activities of the
follistatin-3 polypeptide or portions thereof. Also especially
preferred in this regard are conservative substitutions.
Most highly preferred are nucleic acid molecules encoding the
mature protein having the amino acid sequence shown in SEQ ID NO:2
or the mature follistatin-3 amino acid sequence encoded by the
deposited cDNA clone.
Further embodiment include an isolated nucleic acid molecule
comprising a polynucleotide having a nucleotide sequence a least
90% identical, and more preferably at least 95%, 96%, 97%, 98% or
99% identical to a polynucleotide selected from the group
consisting of (a) a nucleotide Sequence encoding the follistatin-3
polypeptide having the complete amino acid sequence in SEQ ID NO:2
(i.e., positions -26 to 237 of SEQ ID NO:2); (b) a nucleotide
sequence encoding the follistatin-3 polypeptide having the complete
amino acid sequence in SEQ ID NO:2 excepting the N-terminal
methionine (i.e., positions -25 to 237 of SEQ ID NO:2); (C) a
nucleotide sequence encoding the predicted mature follistatin-3
polypeptide having the amino acid sequence at positions 1 to 237 in
SEQ ID NO:2; (d) a nucleotide sequence encoding the follistatin-3
polypeptide having the complete amino acid sequence encoded by the
cDNA clone contained in ATCC.RTM. deposit No. 209197; (e) a
nucleotide sequence encoding the follistatin-3 polypeptide having
the complete amino acid sequence excepting the N-terminal
methionine encoded by the cDNA clone contained in ATCC.RTM. Deposit
No. 209197; (1) a nucleotide sequence encoding the mature
follistatin-3 polypeptide having the amino acid sequence encoded by
the cDNA clone contained in ATCC.RTM. Deposit No. 209197; and (g) a
nucleotide sequence complementary to any of the nucleotide
sequences in (a), (b), (c), (d), (e) or (f) above.
Further embodiments of the invention include isolated nucleic acid
molecules that comprise a polynucleotide having a nucleotide
sequence at least 90% identical, and more preferably at least 95%,
96%, 97%, 98% or 99% identical, to any of the nucleotide sequences
in (a), (b), (c), (d), (e), (f) or (g), above, or a polynucleotide
which hybridizes under stringent hybridization conditions to a
polynucleotide in (a), (b), (c), (d), (e), (f) or (g), above. This
polynucleotide which hybridizes does not hybridize under stringent
hybridization conditions to a polynucleotide having a nucleotide
sequence consisting of only A residues or of only T residues. An
additional nucleic acid embodiment of the invention relates to an
isolated nucleic acid molecule comprising a polynucleotide which
encodes the amino acid sequence of an epitope-bearing portion of a
follistatin-3 polypeptide having an amino acid sequence in (a),
(b), (c), (d), (e) or (f), above. A further nucleic acid embodiment
of the invention relates to an isolated nucleic acid molecule
comprising a polynucleotide which encodes the amino acid sequence
of a follistatin-3 polypeptide having an amino acid sequence which
contains at least one conservative amino acid substitution, but not
more than 50 conservative amino acid substitutions, even more
preferably, not more than 40 conservative amino acid substitutions,
still more preferably not more than 30 conservative amino acid
substitutions, and still even more preferably not more than 20
conservative amino acid substitutions. Of course, in order of
ever-increasing preference, it is highly preferable for a
polynucleotide which encodes the amino acid sequence of a
follistatin-3 polypeptide to have an amino acid sequence which
contains not more than 7-10, 5-10, 3-7, 3-5, 2-5, 1-5, 1-3, 10, 9,
8, 7, 6, 5, 4, 3, 2 or 1 conservative amino acid substitutions.
The present invention also relates to recombinant vectors, which
include the isolated nucleic acid molecules of the present
invention, and to host cells containing the recombinant vectors, as
well as to methods of making such vectors and host cells and for
using them for production of follistatin-3 polypeptides or peptides
by recombinant techniques.
By a polynucleotide having a nucleotide sequence at least, for
example, 95% "identical" to a reference nucleotide sequence
encoding a follistatin-3 polypeptide is intended that the
nucleotide sequence of the polynucleotide is identical to the
reference sequence except that the polynucleotide sequence may
include up to five point mutations per each 100 nucleotides of the
reference nucleotide sequences encoding the follistatin-3
polypeptides. In other words, to obtain a polynucleotide having a
nucleotide sequence at least 95% identical to a reference
nucleotide sequence, up to 5% of the nucleotides in the reference
sequence may be deleted or substituted with another nucleotide, or
a number of nucleotides up to 5% of the total nucleotides in the
reference sequence may be inserted into the reference sequence.
These mutations of the reference sequence may occur at the 5' or 3'
terminal positions of the reference nucleotide sequence or anywhere
between those terminal positions, interspersed either individually
among nucleotides in the reference sequence or in one or more
contiguous groups within the reference sequence.
As a practical matter, whether any particular nucleic acid molecule
is at least 90%, 95%, 96%, 97%, 98% or 99% identical to, for
instance, the nucleotide sequence shown in FIGS. 1A, 1B, and 1C, or
to the nucleotides sequence of the deposited cDNA clone can be
determined conventionally using known computer programs such as the
Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for
Unix, Genetics Computer Group, University Research Park, 575
Science Drive, Madison, Wis. 53711). Bestfit uses the local
homology algorithm of Smith and Waterman to find the best segment
of homology between two sequences (Advances in Applied Mathematics
2:482-489 (1981)). When using Bestfit or any other sequence
alignment program to determine whether a particular sequence is,
for instance, 95% identical to a reference sequence according to
the present invention, the parameters are set, of course, such that
the percentage of identity is calculated over the full length of
the reference nucleotide sequence and that gaps in homology of up
to 5% of the total number of nucleotides in the reference sequence
are allowed. A preferred method for determing the best overall
match between a query sequence (a sequence of the present
invention) and a subject sequence, also referred to as a global
sequence alignment, can be determined using the FASTDB computer
program based on the algorithm of Brutlag and colleagues (Comp.
App. Biosci. 6:237-245 (1990)). In a sequence alignment the query
and subject sequences are both DNA sequences. An RNA sequence can
be compared by converting U's to T's. The result of said global
sequence alignment is in percent identity. Preferred parameters
used in a FASTDB alignment of DNA sequences to calculate percent
identiy are: Matrix=Unitary, k-tuple=4, Mismatch Penalty=1, Joining
Penalty=30, Randomization Group Length=0, Cutoff Score=1, Gap
Penalty=5, Gap Size Penalty 0.05, Window Size=500 or the length of
the subject nucleotide sequence, whichever is shorter.
If the subject sequence is shorter than the query sequence because
of 5' or 3' deletions, not because of internal deletions, a manual
correction must be made to the results. This is because the FASTDB
program does not account for 5' and 3' truncations of the subject
sequence when calculating percent identity. For subject sequences
truncated at the 5' or 3' ends, relative to the the query sequence,
the percent identity is corrected by calculating the number of
bases of the query sequence that are 5' and 3' of the subject
sequence, which are not matched/aligned, as a percent of the total
bases of the query sequence. Whether a nucleotide is
matched/aligned is determined by results of the FASTDB sequence
alignment. This percentage is then subtracted from the percent
identity, calculated by the above FASTDB program using the
specified parameters, to arrive at a final percent identity score.
This corrected score is what is used for the purposes of the
present invention. Only bases outside the 5' and 3' bases of the
subject sequence, as displayed by the FASTDB alignment, which are
not matched/aligned with the query sequence, are calculated for the
purposes of manually adjusting the percent identity score;
For example, a 90 base subject sequence is aligned to a 100 base
query sequence to determine percent identity. The deletions occur
at the 5' end of the subject sequence and therefore, the FASTDB
alignment does not show a matched/alignement of the first 10 bases
at 5' end. The 10 unpaired bases represent 10% of the sequence
(number of bases at the 5' and 3' ends not matched/total number of
bases in the query sequence) so 10% is subtracted from the percent
identity score calculated by the FASTDB program. If the remaining
90 bases were perfectly matched the final percent identity would be
90%. In another example, a 90 base subject sequence is compared
with a 100 base query sequence. This time the deletions are
internal deletions so that there are no bases on the 5' or 3' of
the subject sequence which are not matched/aligned with the query.
In this case the percent identity calculated by FASTDB is not
manually corrected. Once again, only bases 5' and 3' of the subject
sequence which are not matched/aligned with the query sequence are
manually corrected for. No other manual corrections are to made for
the purposes of the present invention.
The present application is directed to nucleic acid molecules at
least 90%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid
sequence shown in FIGS. 1A, 1B, and 1C (SEQ ID NO:1) or to the
nucleic acid sequence of the deposited cDNA, irrespective of
whether they encode a polypeptide having follistatin-3 activity.
This is because even where a particular nucleic acid molecule does
not encode a polypeptide having follistatin-3 activity, one of
skill in the art would still know how to use the nucleic acid
molecule, for instance, as a hybridization probe or a polymerase
chain reaction (PCR) primer. Uses of the nucleic acid molecules of
the present invention that do not encode a polypeptide having
follistatin-3 activity include, inter alia, (1) isolating the
follistatin-3 gene or allelic variants thereof in a cDNA library;
(2) in situ hybridization (e.g., "FISH") to metaphase chromosomal
spreads to provide precise chromosomal location of the
follistatin-3 gene, as described by Verma and colleagues (Human
Chromosomes: A Manual of Basic Techniques, Pergamon Press, New
York. (1988)); and Northern Blot analysis for detecting
follistatin-3 mRNA expression in specific tissues.
Preferred, however, are nucleic acid molecules having sequences at
least 90%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid
sequence shown in FIGS. 1A, 1B, and 1C (SEQ ID NO:1) or to the
nucleic acid sequence of the deposited cDNA which do, in fact,
encode a polypeptide having follistatin-3 activity. By "a
polypeptide having follistatin-3 activity" is intended polypeptides
exhibiting activity similar, but not necessarily identical, to an
activity of the mature follistatin-3 polypeptide of the invention,
as measured in a particular biological assay. For example, the
follistatin-3 polypeptide of the present invention-inhibits the
binding of activin to the activin receptor. An activin
receptor-binding inhibition assay is described by Hashimoto and
colleagues (J. Biol. Chem. 272:13835-13842 (1997)). Briefly, the
assay involves culturing rat pituitary cells (5.times.10.sup.5
cells) in 24-well plates in the presence of [.sup.125 I]-activin A
(40 ng/mL; activin A is labeled using the chloramine-T method as
described by Hasegawa and coworkers (Endocrinol. Japan 33:645-654
(1986)) and follistatin-3 or a mutein thereof (200 .mu.g/mL). A
baseline of activin-binding is determined by affinity cross-linking
[.sup.125 I]-activin A to the pituitary cells using the
bifunctional chemical cross-linker disuccinimidyl suberate (DSS) in
the absence of follistatin-3. Cross-linking is achieved by washing
cells once with binding buffer (DMEM containing 25 mM HEPES (pH
7.4) and 0.2% bovine serum albumen) and incubating on ice for 2 h
with 40 ng/mL [.sup.125 I]-activin A in the binding buffer.
Following incubation, cells are washed 3 times with ice-cold PBS
and incubated in PBS containing 1 mM DSS for 20 min on ice. The
reaction is then quenched with PBS. The cells are removed from the
culture dish by scraping, rinsed with a Tris solution (20 mM
Tris-HCl (pH 7.2) containing 2 mM EDTA, 5 mM benzamidine, 2 mM
phenylmethylsulfonyl fluoride (PMSF), 2 mM N-ethylaleimide, and 2
mM diisopropyl fluorophosphate), centrifuged, and resuspended in
solubilization buffer (50 mM Tris-HCl (pH 7.2) containing 150 mM
NaCl, 2 mM EDTA, 5 mM benzamidine, 2 mM PMSF, 2 mM N-ethylaleimide,
2 mM diisopropyl fluorophosphate, 1% Triton X-100, and 10%
glycerol), and stirred gently on ice for 1 h. The cell lysates are
introduced into 2% SDS and boiled at 100.degree. C. for 10 min. The
resulting affinity-labeled lysates are then subject to SDS-PAGE
(7.5 or 8% gels). Following SDS-PAGE, gels are fixed, stained with
0.25% Coomassie Brilliant Blue R-250, destained, air-dried, and
then visualized by autoradiography. Inhibition of activin binding
of the activin receptor is analyzed in samples with which
follistatin-3 or a mutein thereof (200 .mu.g/mL) are incubated with
labeled activin in the binding buffer incubation described above.
The degree to which the formation of affinity cross-linked
activin/activin receptor complexes is decreased correlates with the
ability of follistatin-3 or a mutein thereof to bind to labeled
activin protein. As such, the relative binding affinity of activin
for its receptor versus follistatin-3 or a mutein thereof can be
quantitated. Such activity is useful for regulating the effective
amount of activin present in a given system.
Follistatin-3 binds to activin in a dose-dependent manner in the
above-described assay. While polypeptides of the invention need not
demonstrate dose-dependent follistatin-3 activity in a bioassay, it
is preferred that, by "a polypeptide having follistatin-3 activity"
is meant a polypeptide that also exhibits any of the same binding
activities in the above-described assays in a dose-dependent
manner. Thus, although the degree of dose-dependent activity need
not be identical to that of the follistatin-3, most preferably, "a
polypeptide having follistatin-3 activity" will exhibit
substantially similar dose-dependence in a given activity as
compared to the follistatin-3 (i.e., the candidate polypeptide will
exhibit greater activity or not more than about 25-fold less and,
preferably, not more than about tenfold less activity relative to
the reference follistatin-3).
Like follistatin-1, follistatin-3 inhibits the secretion of FSH. An
assay for measuring the suppression of spontaneous FSH release from
primary cultured rat pituitary cells is well known in the art
(Hasegawa, Y., et al., Endocrinol. Jpn. 33:645-654 (1986)).
Briefly, freshly isolated pituitary cells are suspended in DMEM
containing gentamicin (35 .mu.g/mL), fungizone (1 .mu.g/mL), 0.05%
glutamine, 0.1% sodium bicarbonate, 10% horse serum, and 2.5% fetal
bovine serum at a density of 3.times.10.sup.5 cells/mL, and plated
in 96-well culture plates (6.times.10.sup.4 cells/0.2 mL/well).
Various amounts (0.1-100 ng/mL) of follistatin-3 are then added to
the culture medium. After culturing for 3 days at 37.degree. C. (5%
CO.sub.2), cultured media are assayed for quantity of secreted FSH
by a double antibody RIA method using an RIA kit and plotted as FSH
Secreted (ng/mL/72 h) versus Protein Added (ng/mL).
Of course, due to the degeneracy of the genetic code, one of
ordinary skill in the art will immediately recognize that a large
number of the nucleic acid molecules having a sequence at least
90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid
sequence of the deposited cDNA or the nucleic acid sequence shown
in FIGS. 1A, 1B, and 1C (SEQ ID NO:1) will encode a polypeptide
"having follistatin-3 activity." In fact, since degenerate variants
of these nucleotide sequences all encode the same polypeptide, this
will be clear to the skilled artisan even without performing the
above described comparison assay. It will be further recognized in
the art that, for such nucleic acid molecules that are not
degenerate variants, a reasonable number will also encode a
polypeptide having follistatin-3 activity. This is because the
skilled artisan is fully aware of amino acid substitutions that are
either less likely or not likely to significantly effect protein
function (e.g., replacing one aliphatic amino acid with a second
aliphatic amino acid), as further described below.
Vectors and Host Cells
While the follistatin-3 polypeptides (including fragments, variants
derivatives, and analogs) of the invention can be chemically
synthesized (e.g., see Creighton, 1983, Proteins: Structures and
Molecular Principles, W.H. Freeman & Co., New York),
follistatin-3 polypeptides may advantageously be produced by
recombinant DNA technology using techniques well known in the art
for expressing gene sequences and/or nucleic acid coding sequences.
Such methods can be used to construct expression vectors containing
the polynucleotides of the invention and appropriate
transcriptional and translational control signals. These methods
include, for example, in vitro recombinant DNA techniques,
synthetic techniques, and in vivo genetic recombination. See, for
example, the techniques described in Sambrook et al., 1989, supra;
Ausubel et al., 1989, supra; Caruthers et al., 1980, Nuc. Acids
Res. Symp. Ser. 7:215-233; Crea and Horn, 1980, Nuc. Acids Res.
9(10):2331; Matteucci and Caruthers, 1980, Tetrahedron Letters
21:719; and Chow and Kempe, 1981, Nuc. Acids Res. 9(12):2807-2817.
Alternatively, RNA capable of producing follistatin-3 sequences may
be chemically synthesized using, for example, synthesizers. See,
for example, the techniques described in "Oligonucleotide
Synthesis", 1984, Gait, M. J. ed., IRL Press, Oxford, which is
incorporated by reference herein in its entirety.
Thus, in one embodiment, the present invention relates to vectors
which include the isolated DNA molecules (i.e., polynucleotides) of
the present invention, host cells which are genetically engineered
with the recombinant vectors, and the production of follistatin-3
polypeptides or fragments thereof by recombinant techniques using
these host cells or host cells that have otherwise been genetically
engineered using techniques known in art to express a polypeptide
of the invention. The vector may be, for example, a phage, plasmid,
viral or retroviral vector. Retroviral vectors may be replication
competent or replication defective. In the latter case, viral
propagation generally will occur only in complementing host
cells.
The polynucleotides may be joined to a vector containing a
selectable marker for propagation in a host. Generally, a plasmid
vector is introduced in a precipitate, such as a calcium phosphate
precipitate, or in a complex with a charged lipid. If the vector is
a virus, it may be packaged in vitro using an appropriate packaging
cell line and then transduced into host cells.
In one embodiment, the polynucleotide of the invention is
operatively associated with an appropriate heterologous regulatory
element (e.g., a promoter or enhancer or both), such as the phage
lambda PL promoter, the E. coli lac, trp, phoA and tac promoters,
the SV40 early and late promoters and promoters of retroviral LTRs,
to name a few. Other suitable promoters will be known to the
skilled artisan.
In embodiments in which vectors contain expression constructs,
these constructs will further contain sites for transcription
initiation, termination and, in the transcribed region, a ribosome
binding site for translation. The coding portion of the transcripts
expressed by the constructs will preferably include a translation
initiating codon at the beginning and a termination codon (UAA, UGA
or UAG) appropriately positioned at the end of the polypeptide to
be translated.
As indicated, the expression vectors will preferably include at
least one selectable marker. Such markers include dihydrofolate
reductase, G418 or neomycin resistance for eukaryotic cell culture
and tetracycline, kanamycin or ampicillin resistance genes for
culturing in E. coli and other bacteria. Representative examples of
appropriate hosts include, but are not limited to, bacterial cells,
such as E. coli, Streptomyces and Salmonella typhimurium cells;
fungal cells, such as yeast cells; insect cells such as Drosophila
S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, 293 and
Bowes melanoma cells; and plant cells. Appropriate culture mediums
and conditions for the above-described host cells are known in the
art.
Vectors preferred for use in bacteria include pHE4-5, pQE70, pQE60
and pQE-9 (QIAGEN, Inc., supra); pBS vectors, Phagescript vectors,
Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A (Stratagene); and
ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia). Among
preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXT1, and
pSG (Stratagene); and pSVK3, pBPV, pMSG and pSVL (Pharmacia). Other
suitable vectors will be readily apparent to the skilled
artisan.
Introduction of the construct into the host cell can be effected by
calcium phosphate transfection, DEAE-dextran mediated transfection,
cationic lipid-mediated transfection, electroporation,
transduction, infection or other methods. Such methods are
described in many standard laboratory manuals (for example, Davis,
et al., Basic Methods In Molecular Biology (1986)).
In addition to encompassing host cells containing the vector
constructs discussed herein, the invention also encompasses
primary, secondary, and immortalized host cells of vertebrate
origin, particularly those of mammalian origin, that have been
engineered to delete or replace endogenous genetic material (e.g.,
foulistatin-3 coding sequence), and/or to include genetic material
(e.g. heterologous polynucleotide sequences) that is operably
associated with follistatin-3 polynucleotides of the invention, and
which activates, alters, and/or amplifies endogenous follistatin-3
polynucleotides. For example, techniques known in the art may be
used to operably associate heterologous control regions (e.g.
promoter and/or enhancer) and endogenous follistatin-3
polynucleotide sequences via homologous recombination (see, e.g.
U.S. Pat. No. 5,641,670, issued Jun. 24, 1997; International
Publication No. WO 96/29411, published Sep. 26, 1996; International
Publication No. WO 94/12650, published Aug. 4, 1994; Koller et al.,
Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); and Zijlstra, et
al., Nature 342:435-438 (1989), the disclosures of each of which
are hereby incorporated by reference in their entireties).
The polypeptide may be expressed in a modified form, such as a
fusion protein, and may include not only secretion signals, but
also additional heterologous functional regions. For instance, a
region of additional amino acids, particularly charged amino acids,
may be added to the N-terminus of the polypeptide to improve
stability and persistence in the host cell, during purification, or
during subsequent handling and storage. Also, peptide moieties may
be added to the polypeptide to facilitate purification. Such
regions may be removed prior to final preparation of the
polypeptide. The addition of peptide moieties to polypeptides to
engender secretion or excretion, to improve stability and to
facilitate purification, among others, are familiar and routine
techniques in the art. A preferred fusion protein comprises a
heterologous region from immunoglobulin that is useful to stabilize
and purify proteins. For example, EP-A-O 464 533 (Canadian
counterpart 2045869) discloses fusion proteins comprising various
portions of constant region of immunoglobulin molecules together
with another human protein or part thereof. In many cases, the Fc
part in a fusion protein is thoroughly advantageous for use in
therapy and diagnosis and thus results, for example, in improved
pharmacokinetic properties (EP-A 0232 262). On the other hand, for
some uses it would be desirable to be able to delete the Fc part
after the fusion protein has been expressed, detected and purified
in the advantageous manner described. This is the case when Fc
portion proves to be a hindrance to use in therapy and diagnosis,
for example when the fusion protein is to be used as antigen for
immunizations. In drug discovery, for example, human proteins, such
as hIL-5, have been fused with Fc portions for the purpose of
high-throughput screening assays to identify antagonists of hIL-5
(Bennett, D., et al., J. Molecular Recognition 8:52-58 (1995);
Johanson, K., et al., J. Biol. Chem. 270:9459-9471 (1995)).
The follistatin-3 polypeptides can be recovered and purified from
recombinant cell cultures by well-known methods including ammonium
sulfate or ethanol precipitation, acid extraction, anion or cation
exchange chromatography, phosphocellulose chromatography,
hydrophobic interaction chromatography, affinity chromatography,
hydroxylapatite chromatography and lectin chromatography. Most
preferably, high performance liquid chromatography ("HPLC") is
employed for purification. Polypeptides of the present invention
include: products purified from natural sources, including bodily
fluids, tissues and cells, whether directly isolated or cultured;
products of chemical synthetic procedures; and products produced by
recombinant techniques from a prokaryotic or eukaryotic host,
including, for example, bacterial, yeast, higher plant, insect and
mammalian cells. Depending upon the host employed in a recombinant
production procedure, the polypeptides of the present invention may
be glycosylated or may be non-glycosylated. In addition,
polypeptides of the invention may also include an initial modified
methionine residue, in some cases as a result of host-mediated
processes. Thus, it is well known in the art that the N-terminal
methionine encoded by the translation initiation codon generally is
removed with high efficiency from any protein after translation in
all eukaryotic cells. While the N-terminal methionine on most
proteins also is efficiently removed in most prokaryotes, for some
proteins this prokaryotic removal process is inefficient, depending
on the nature of the amino acid to which the N-terminal methionine
is covalently linked.
Included-within the scope of the invention are follistatin-3
polypeptides (including fragments, variants, derivatives and
analogs) which are differentially modified during or after
translation, e.g., by glycosylation, acetylation, phosphorylation,
amidation, derivatization by known protecting/blocking groups,
proteolytic cleavage, linkage to an antibody molecule or other
cellular ligand, etc. Any of numerous chemical modifications may be
carried out by known techniques, including, but not limited to,
specific chemical cleavage by cyanogen bromide, trypsin,
chymotrypsin, papain, V8 protease, NaBH4; acetylation, formylation,
oxidation, reduction; metabolic synthesis in the presence of
tunicamycin; etc. In a specific embodiment, the compositions of the
invention are conjugated to other molecules to increase their
water-solubility (e.g., polyethylene glycol), half-life, or ability
to bind targeted tissue.
Polypeptides and Fragments
The invention further provides an isolated follistatin-3
polypeptide having the amino acid sequence encoded by the deposited
cDNA, or the amino acid sequence in SEQ ID NO:2, or a peptide or
polypeptide comprising fragment (i.e., a portion) of the above
polypeptides.
The polypeptides and polynucleotides of the present invention are
preferably provided in an isolated form, and preferably are
purified to a point within the range of near complete (e.g.,
>90% pure) to complete (e.g., >99% pure) homogeneity. The
term "isolated" means that the material is removed from its
original environment (e.g., the natural environment if it is
naturally occurring). For example, a naturally-occurring
polynucleotide or polypeptide present in a living animal is not
isolated, but the same polynucleotide or polypeptide, separated
from some or all of the coexisting materials in the natural system,
is isolated. Also intended as an "isolated polypeptide" are
polypeptides that have been purified partially or substantially
from a recombinant host cell. For example, a recombinantly produced
version of a follistatin-3 polypeptide can be substantially
purified by the one-step method described by Smith and Johnson
(Gene 67:31-40 (1988)). Such polynucleotides could be part of a
vector and/or such polynucleotides or polypeptides could be part of
a composition, and still be isolated in that such vector or
composition is not part of its natural environment. Isolated
polypeptides and polynucleotides according to the present invention
also include such molecules produced naturally or synthetically.
Polypeptides and polynucleotides of the invention also can be
purified from natural or-recombinant sources using
anti-follistatin-3 antibodies of the invention which may routinely
be generated and utilized using methods known in the art.
The present invention also encompasses fragments of the
above-described follistatin-3 polypeptides. Polypeptide fragments
of the present invention include polypeptides comprising an amino
acid sequence contained in SEQ ID NO:2, encoded by the cDNA
contained in the deposited clone, or encoded by nucleic acids which
hybridize (e.g., under stringent hybridization conditions) to the
nucleotide sequence contained in the deposited clones, that shown
in FIGS. 1A, 1B, and 1C (SEQ ID NO:1), or the complementary strand
thereto.
The polynucleotide fragments of the invention encode a polypeptide
which demonstrates a functional activity. By a polypeptide
demonstrating "functional activity" is meant, a polypeptide capable
of displaying one or more known functional activities associated
with a complete, mature or active form of the follistatin-3
polypeptide. Such functional activities include, but are not
limited to, biological activity ((e.g., modulating the follicle
stimulating hormone (FSH) synthetic pathway, increasing estradiol
production, binding activin, stimulating gonadotropin biosynthesis
and secretion, regulating of ovarian and placental steroidogenesis,
and oocyte and spermatogonial maturation factor)), antigenicity
[ability to bind (or compete with a follistatin-3 polypeptide for
binding) to an anti-follistatin-3 antibody], immunogenicity
(ability to generate antibody which binds to a follistatin-3
polypeptide), the ability to form polymers with other follistatin-3
or inhibin or TGF-b polypeptides, and ability to bind to a receptor
or ligand for a follistatin-3 polypeptide (e.g., an activin).
Polypeptide fragments may be "free-standing" or comprised within a
larger polypeptide of which the fragment forms a part or region,
most preferably as a single continuous region. Representative
examples of polypeptide fragments of the invention, included, for
example, fragments that comprise or alternatively, consist of, from
about amino acid residues, 1 to 20, 21 to 40, 41 to 60, 61 to 83,
84 to 100, 101 to 120, 121 to 140, 141 to 160, 161 to 180, 181 to
200, 201 to 220, 201 to 224, 210 to 231, 221 to 240, or 241 to 263
of SEQ ID NO:2. Moreover, polypeptide fragments can be at least
about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150,
160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260 amino acids
in length. In this context "about" includes the particularly
recited ranges, larger or smaller by several (i.e. 5, 4, 3, 2 or 1)
amino acids, at either extreme or at both extremes.
In other embodiments, the fragments or polypeptides of the
invention (i.e., those described herein) are not larger than 250,
225, 200, 185, 175, 170, 165, 160, 155, 150, 145, 140, 135, 130,
125, 120, 115, 110, 105, 100, 90, 80, 75, 60, 50, 40, 30 or 25
amino acids residues in length.
Additional embodiments encompass polypeptide fragments comprising
one, two, three, four, five, or more functional attributes of
follistatin-3 polypeptides of the invention, such as, one or more
Garnier-Robson alpha-regions, beta-regions, turn-regions, and
coil-regions, Chou-Fasman alpha-regions, beta-regions, and
coil-regions, Kyte-Doolittle hydrophilic regions and hydrophobic
regions, Eisenberg alpha- and beta-amphipathic regions,
Karplus-Schulz flexible regions, Emini surface-forming regions and
Jameson-Wolf regions of high antigenic index, or any combination
thereof, as disclosed in FIG. 3 and in Table I as described
herein.
Preferred polypeptides of the invention comprise, or alternatively,
consist of amino acid residues 7-16, 34-45, 78-86, 91-100, 108-122,
131-145, 156-169, 184-192, and/or 196-210 of SEQ ID NO:2.
Polynucleotides encoding these polypeptides are also encompassed by
the invention, as are polynucleotides that hybridize to the
complementary strand of these encoding polynucleotides under high
stringency conditions (e.g., as described herein) and polypeptides
encoded by these hybridizing polynucleotides.
In specific embodiments, polypeptide fragments of the invention
comprise, or alternatively, consist of, amino acid residues Leu-14
to Ala-20, Ser-46 to Ile-55, Gly-88 to Pro-97, Gly-113 to Leu-133,
Arg-138 to Glu-146, Pro-177 to Thr-191, and/or Gly-219 to Val-237
of SEQ ID NO:2. These polypeptide fragments have been determined to
bear antigenic epitopes of the follistatin-3 by the analysis of the
Jameson-Wolf antigenic index, as shown in FIG. 3 and Table I,
above. Polynucleotides encoding these polypeptides are also
encompassed by the invention, as are polynucleotides that hybridize
to the complementary strand of these encoding polynucleotides under
high stringency conditions (e.g., as described herein) and
polypeptides encoded by these hybridizing polynucleotides.
As described in detail below, the polypeptides of the present
invention can also be used to raise polyclonal and monoclonal
antibodies, which are useful in assays for detecting follistatin-3
expression as described below or as agonists and antagonists
capable of enhancing or inhibiting follistatin-3 function. Further,
such polypeptides can be used in the yeast two-hybrid system to
"capture" follistatin-3 binding proteins which are also candidate
agonists and antagonists according to the present invention. The
yeast two hybrid system is described by Fields and Song (Nature
340:245-246 (1989)).
In another aspect, the invention provides a peptide or polypeptide
comprising an epitope-bearing portion of a polypeptide of the
invention. The epitope of this polypeptide portion is an
immunogenic or antigenic epitope of a polypeptide of the invention.
An "immunogenic epitope" is defined as a part of a protein that
elicits an antibody response when the whole protein is the
immunogen. On the other hand, a region of a protein molecule to
which an antibody can bind is defined as an "antigenic epitope".
The number of immunogenic epitopes of a protein generally is less
than the number of antigenic epitopes (see, for instance, Geysen,
et al., Proc. Natl. Acad. Sci. USA 81:3998-4002 (1983)).
As to the selection of peptides or polypeptides bearing an
antigenic epitope (i.e., that contain a region of a protein
molecule to which an antibody can bind), it is well known in that
art that relatively short synthetic peptides that mimic part of a
protein sequence are routinely capable of eliciting an antiserum
that reacts with the partially mimicked protein (see, for instance,
Sutcliffe, J. G., et al., Science 219:660-666 (1983)). Peptides
capable of eliciting protein-reactive sera are frequently
represented in the primary sequence of a protein, can be
characterized by a set of simple chemical rules, and are confined
neither to immunodominant regions of intact proteins (i.e.,
immunogenic epitopes) nor to the amino or carboxyl terminals.
Antigenic epitope-bearing peptides and polypeptides of the
invention are therefore useful to raise antibodies, including
monoclonal antibodies, that bind specifically to a polypeptide of
the invention (see, for instance, Wilson, et al., Cell 37:767-778
(1984)).
Antigenic epitope-bearing peptides and polypeptides of the
invention preferably contain a sequence of at least seven, more
preferably at least nine and most preferably between about 15 to
about 30 amino acids contained within the amino acid sequence of a
polypeptide of the invention. Non-limiting examples of antigenic
polypeptides or peptides that can be used to generate
follistatin-3-specific antibodies include: a polypeptide comprising
amino acid residues: Leu-14 to Ala-20, Ser-46 to Ile-55, Gly-88 to
Pro-97, Gly-113 to Leu-133, Arg-138 to Glu-146, Pro-177 to Thr-191,
and/or Gly-219 to Val-237 of SEQ ID NO:2. These polypeptide
fragments have been determined to bear antigenic epitopes of the
follistatin-3 by the analysis of the Jameson-Wolf antigenic index,
as shown in FIG. 3 and Table I, above.
The epitope-bearing peptides and polypeptides of the invention may
be produced by any conventional means (see, for example, Houghten,
R. A., et al., Proc. Natl. Acad. Sci. USA 82:5131-5135 (1985); and
U.S. Pat. No. 4,631,211 to Houghten, et al. (1986)).
Epitope-bearing peptides and polypeptides of the invention are used
to induce antibodies according to methods well known in the art
(see, for instance, Sutcliffe, et al., supra; Wilson, et al.,
supra; Chow, M., et al., Proc. Natl. Acad. Sci. USA 82:910-914; and
Bittle, F. J., et al., J. Gen. Virol. 66:2347-2354 (1985)).
Immunogenic epitope-bearing peptides of the invention, i.e., those
parts of a protein that elicit an antibody response when the whole
protein is the immunogen, are identified according to methods known
in the art (see, for instance, Geysen, et al., supra). Further
still, U.S. Pat. No. 5,194,392, issued to Geysen, describes a
general method of detecting or determining the sequence of monomers
(amino acids or other compounds) which is a topological equivalent
of the epitope (i.e., a "mimotope") which is complementary to a
particular paratope (antigen binding site) of an antibody of
interest. More generally, U.S. Pat. No. 4,433,092, issued to
Geysen, describes a method of detecting or determining a sequence
of monomers which is a topographical equivalent of a ligand which
is complementary to the ligand binding site of a particular
receptor of interest. Similarly, U.S. Pat. No. 5,480,971, issued to
Houghten and colleagues, on Peralkylated Oligopeptide Mixtures
discloses linear C1-C7-alkyl peralkylated oligopeptides and sets
and libraries of such peptides, as well as methods for using such
oligopeptide sets and libraries for determining the sequence of a
peralkylated oligopeptide that preferentially binds to an acceptor
molecule of interest. Thus, non-peptide analogs of the
epitope-bearing peptides of the invention also can be made
routinely by these methods.
To improve or alter the characteristics of follistatin-3
polypeptides, protein engineering may be employed. Recombinant DNA
technology known to those skilled in the art can be used to create
novel mutant proteins or muteins including single or multiple amino
acid substitutions, deletions, additions or fusion proteins. Such
modified polypeptides can show, e.g., enhanced activity or
increased stability. In addition, they may be purified in higher
yields and show better solubility than the corresponding natural
polypeptide, at least under certain purification and storage
conditions.
For instance, for many proteins, including the extracellular domain
of a membrane associated protein or the mature form(s) of a
secreted protein, it is known in the art that one or more amino
acids may be deleted from the N-terminus or C-terminus without
substantial loss of biological function. For instance, Ron and
colleagues (J. Biol. Chem., 268:2984-2988 (1993)) reported modified
KGF proteins that had heparin binding activity even if 3, 8, or 27
N-terminal amino acid residues were missing. In the present case,
since the protein of the invention is a member of the
inhibin-related polypeptide family, deletions of N-terminal amino
acids up to the cysteine at position 12 of SEQ ID NO:2 may retain
some biological activity such as binding activin or an activin-like
molecule. Polypeptides having further N-terminal deletions
including the cysteine-12 residue in SEQ ID NO:2 would not be
expected to retain such biological activities because it is known
that this residue is likely required for forming a disulfide bridge
to provide structural stability which is needed for protein-protein
interaction and is in the beginning of the conserved domain
required for biological activities.
However, even if deletion of one or more amino acids from the
N-terminus of a protein results in modification of loss of one or
more biological functions of the protein, other functional or
biological activities may still be retained. Thus, the ability of
the shortened protein to induce and/or bind to antibodies which
recognize the complete or mature of the protein generally will be
retained when less than the majority of the residues of the
complete or mature protein are removed from the N-terminus. Whether
a particular polypeptide lacking N-terminal residues of a complete
protein retains such immunologic activities can readily be
determined by routine methods described herein and otherwise known
in the art.
Accordingly, the present invention further provides polypeptides
having one or more residues deleted from the amino terminus of the
amino acid sequence of the follistatin-3 shown in SEQ ID NO:2, up
to the cysteine residue at position number 12, and polynucleotides
encoding such polypeptides. In particular, the present invention
provides polypeptides comprising the amino acid sequence of
residues n.sup.1 -237 of SEQ ID NO:2, where n.sup.1 is an integer
in the range of -26-.sup.12, and 12 is the position of the first
residue from the N-terminus of the complete follistatin-3
polypeptide (shown in SEQ ID NO:2) believed to be required for
activin-binding or activin-like protein-binding activity of the
follistatin-3.
More in particular, the invention provides polynucleotides encoding
polypeptides having the amino acid sequence of residues of -26-237,
-25-237, -24-237, -23-237, -22-237, -21-237, -20-237, -19-237,
-18-237, -17-237, -16-237, -15-237, -14-237, -13-237, -12-237,
-10-237, -9-237, -8-237, -7-237, -6-237, -5-237, -4-237, -3-237,
-2-237, -1-237, 1-237, 2-237, 3-237, 4-237, 5-237, 6-237, 7-237,
8-237, 9-237, 10-237, 11-237, and 12-237 of SEQ ID NO:2.
Polynucleotides encoding these polypeptides also are provided.
Similarly, many examples of biologically functional C-terminal
deletion muteins are known. For instance, Interferon gamma shows up
to ten times higher activities by deleting 8-10 amino acid residues
from the carboxy terminus of the protein (Dobeli, et al., J.
Biotechnology 7:199-216 (1988)). In the present case, since the
protein of the invention is a member of the activin-related
polypeptide family, deletions of C-terminal amino acids up to the
cysteine at position 217 of SEQ ID NO:2 may retain some biological
activity such as binding activin or an activin-like molecule.
Polypeptides having further C-terminal deletions including the
cysteine residue at position 217 of SEQ ID NO:2 would not be
expected to retain such biological activities because it is known
that this residue is likely required for forming a disulfide bridge
to provide structural stability which is needed for protein-protein
interactions and is the beginning of the conserved domain required
for biological activities.
However, even if deletion of one or more amino acids from the
C-terminus of a protein results in modification of loss of one or
more biological functions of the protein, other functional or
biological activities may still be retained. Thus, the ability of
the shortened protein to induce and/or bind to antibodies which
recognize the complete or mature form of the protein generally will
be retained when less than the majority of the residues of the
complete or mature form of the protein are removed from the
C-terminus. Whether a particular polypeptide lacking C-terminal
residues of a complete protein retains such immunologic activities
can readily be determined by routine methods described herein and
otherwise known in the art.
Accordingly, the present invention further provides polypeptides
having one or more residues from the carboxy terminus of the amino
acid sequence of the follistatin-3 shown in SEQ ID NO:2, up to the
cysteine residue at position 217 of SEQ ID NO:2, and
polynucleotides encoding such polypeptides. In particular, the
present invention provides polypeptides having the amino acid
sequence of residues -26-m.sup.1 of the amino acid sequence in SEQ
ID NO:2, where m' is any integer in the range of 217 to 237, and
residue 217 is the position of the first residue from the
C-terminus of the complete follistatin-3 polypeptide (shown in SEQ
ID NO:2) believed to be required for the activin-binding or
activin-like protein-binding of the follistatin-3.
More in particular, the invention provides polynucleotides encoding
polypeptides having the amino acid sequence of residues -26-217,
-26-218, -26-219, -26-220, -26-221, -26-222, -26-223, -26-224,
-26-225, -26-226, -26-227, -26-228, -26-229, -26-230, -26-231,
-26-232, -26-233, -26-234, -26-235, -26-236, and -26-237 of SEQ ID
NO:2. Polynucleotides encoding these polypeptides also are
provided.
The invention also provides polypeptides having one or more amino
acids deleted from both the amino and the carboxyl termini, which
may be described generally as having residues n.sup.1 -m.sup.1 of
SEQ ID NO:2, where n.sup.1 and m.sup.1 are integers as described
above.
Also included are a nucleotide seqaence encoding a polypeptide
consisting of a portion of the complete follistatin-3 amino acid
sequence encoded by the cDNA clone contained in ATCC.RTM. Deposit
No. 209197, where this portion exclude from 1 to abota 37 amino
acids from the amino terminus of the complete amino acid sequence
encoded by tue cDNA clone contained in ATCC.RTM. Depsoit No.
209197, or from 1 to about 20 amino acid from the carboxy terminus,
of any cwnbinarion of the above amino terminal and carboxy terminal
deletion, of the complete ammo acid sequence encoded by the cDNA
clone contdrncd ifl ATCC.RTM. Deposit No. 209197. Polynucleotides
encoaing all of the above deletion mutant polypeptide forms also
are provided.
As mentioned above, even if deletion of one or more amino acids
from the N-terminus of a protein results in modification of loss of
one or more functions of the protein, other functional or
biological activities may still be retained. Thus, the ability of
the shortened follistatin-3 mutein to induce and/or bind to
antibodies which recognize the complete or mature of the protein
generally will be retained when less than the majority of the
residues of the complete or mature protein are removed from the
N-terminus. Whether a particular polypeptide lacking N-terminal
residues of a complete protein retains such immunologic activities
can readily be determined by routine methods described herein and
otherwise known in the art. It is not unlikely that a follistatin-3
mutein with a large number of deleted N-terminal amino acid
residues may retain some biological or immungenic activities. In
fact, peptides composed of as few as six follistatin-3 amino acid
residues may often evoke an immune response.
Accordingly, the present invention further provides polypeptides
having one or more residues deleted from the amino terminus of the
follistatin-3 amino acid sequence shown in SEQ ID NO:2, up to the
glutamic acid residue at position number 258 and polynucleotides
encoding such polypeptides. In particular, the present invention
provides polypeptides comprising the amino acid sequence of
residues n.sup.2 -263 of FIGS. 1A, 1B, and 1C (SEQ ID NO:2), where
n.sup.2 is an integer in the range of 2 to 258, and 259 is the
position of the first residue from the N-terminus of the complete
follistatin-3 polypeptide believed to be required for at least
immunogenic activity of the follistatin-3.
More in particular, the invention provides polynucleotides encoding
polypeptides comprising, or alternatively consisting of, the amino
acid sequence of residues of R-2 to V-263; P-3 to V-263; G-4 to
V-263; A-5 to V-263; P-6 to V-263; G-7 to V-263; P-8 to V-263; L-9
to V-263; W-10 to V-263; P-11 to V-263; L-12 to V-263; P-13 to
V-263; W-14 to V-263; G-15 to V-263; A-16 to V-263; L-17 to V-263;
A-18 to V-263; W-19 to V-263; A-20 to V-263; V-21 to V-263; G-22 to
V-263; F-23 to V-263; V-24 to V-263; S-25 to V-263; S-26 to V-263;
M-27 to V-263; G-28 to V-263; S-29 to V-263; G-30 to V-263; N-31 to
V-263; P-32 to V-263; A-33 to V-263; P-34 to V-263; G-35 to V-263;
G-36 to V-263; V-37 to V-263; C-38 to V-263; W-39 to V-263; L-40 to
V-263; Q-41 to V-263; Q-42 to V-263; G-43 to V-263; Q-44 to V-263;
E-45 to V-263; A-46 to V-263; T-47 to V-263; C-48 to V-263; S-49 to
V-263; L-50 to V-263; V-51 to V-263; L-52 to V-263; Q-53 to V-263;
T-54 to V-263; D-55 to V-263; V-56 to V-263; T-57 to V-263; R-58 to
V-263; A-59 to V-263; E-60 to V-263; C-61 to V-263; C-62 to V-263;
A-63 to V-263; S-64 to V-263; G-65 to V-263; N-66 to V-263; I-67 to
V-263; D-68 to V-263; T-69 to V-263; A-70 to V-263; W-71 to V-263;
S-72 to V-263; N-73 to V-263; L-74 to V-263; T-75 to V-263; H-76 to
V-263; P-77 to V-263; G-78 to V-263; N-79 to V-263; K-80 to V-263;
I-81 to V-263; N-82 to V-263; L-83 to V-263; L-84 to V-263; G-85 to
V-263; F-86 to V-263; L-87 to V-263; G-88 to V-263; L-89 to V-263;
V-90 to V-263; H-91 to V-263; C-92 to V-263; L-93 to V-263; P-94 to
V-263; C-95 to V-263; K-96 to V-263; D-97 to V-263; S-98 to V-263;
C-99 to V-263; D-100 to V-263; G-101 to V-263; V-102 to V-263;
E-103 to V-263; C-104 to V-263; G-105 to V-263; P-106 to V-263;
G-107 to V-263; K-108 to V-263; A-109 to V-263; C-110 to V-263;
R-111 to V-263; M-112 to V-263; L-113 to V-263; G-114 to V-263;
G-115 to V-263; R-116 to V-263; P-117 to V-263; R-118 to V-263;
C-119 to V-263; E-120 to V-263; C-121 to V-263; A-122 to V-263;
P-123 to V-263; D-124 to V-263; C-125 to V-263; S-126 to V-263;
G-127 to V-263; L-128 to V-263; P-129 to V-263; A-130 to V-263;
R-131 to V-263; L-132 to V-263; Q-133 to V-263; V-134 to V-263;
C-135 to V-263; G-136 to V-263; S-137 to V-263; D-138 to V-263;
G-139 to V-263; A-140 to V-263; T-141 to V-263; Y-142 to V-263;
R-143 to V-263; D-144 to V-263; E-145 to V-263; C-146 to V-263;
E-147 to V-263; L-148 to V-263; R-149 to V-263; A-150 to V-263;
A-151 to V-263; R-152 to V-263; C-153 to V-263; R-154 to V-263;
G-155 to V-263; H-156 to V-263; P-157 to V-263; D-158 to V-263;
L-159 to V-263; S-160 to V-263; V-161 to V-263; M-162 to V-263;
Y-163 to V-263; R-164 to V-263; G-165 to V-263; R-166 to V-263;
C-167 to V-263; R-168 to V-263; K-169 to V-263; S-170 to V-263;
C-171 to V-263; E-172 to V-263; H-173 to V-263; V-174 to V-263;
V-175 to V-263; C-176 to V-263; P-177 to V-263; R-178 to V-263;
P-179 to V-263; Q-180 to V-263; S-181 to V-263; C-182 to V-263;
V-183 to V-263; V-184 to V-263; D-185 to V-263; Q-186 to V-263;
T-187 to V-263; G-188 to V-263; S-189 to V-263; A-190 to V-263;
H-191 to V-263; C-192 to V-263; V-193 to V-263; V-194 to V-263;
C-195 to V-263; R-196 to V-263; A-197 to V-263; A-198 to V-263;
P-199 to V-263; C-200 to V-263; P-201 to V-263; V-202 to V-263;
P-203 to V-263; S-204 to V-263; S-205 to V-263; P-206 to V-263;
G-207 to V-263; Q-208 to V-263; E-209 to V-263; L-210 to V-263;
C-211 to V-263; G-212 to V-263; N-213 to V-263; N-214 to V-263;
N-215 to V-263; V-216 to V-263; T-217 to V-263; Y-218 to V-263;
I-219 to V-263; S-220 to V-263; S-221 to V-263; C-222 to V-263;
H-223 to V-263; M-224 to V-263; R-225 to V-263; Q-226 to V-263;
A-227 to V-263; T-228 to V-263; C-229 to V-263; F-230 to V-263;
L-231 to V-263; G-232 to V-263; R-233 to V-263; S-234 to V-263;
I-235 to V-263; G-236 to V-263; V-237 to V-263; R-238 to V-263;
H-239 to V-263; A-240 to V-263; G-241 to V-263; S-242 to V-263;
C-243 to V-263; A-244 to V-263; G-245 to V-263; T-246 to V-263;
P-247 to V-263; E-248 to V-263; E-249 to V-263; P-250 to V-263;
P-251 to V-263; G-252 to V-263; G-253 to V-263; E-254 to V-263;
S-255 to V-263; A-256 to V-263; E-257 to V-263; and E-258 to V-263
of the follistatin-3 amino acid sequence shown in FIGS. 1A, 1B, and
1C (which is identical to the sequence shown as SEQ ID NO:2, with
the exception that the amino acid residues in FIGS. 1A, 1B, and 1C
are numbered consecutively from 1 through 263 from the N-terminus
to the C-terminus, while the amino acid residues in SEQ ID NO:2 are
numbered consecutively from -26 through 237 to reflect the position
of the predicted signal peptide). Polynucleotides encoding these
polypeptides are also encompassed by the invention.
Also as mentioned above, even if deletion of one or more amino
acids from the C-terminus of a protein results in modification of
loss of one or more biological functions of the protein, other
functional or biological activities may still be retained. Thus,
the ability of the shortened follistatin-3 mutein to induce and/or
bind to antibodies which recognize the complete or mature of the
protein generally will be retained when less than the majority of
the residues of the complete or mature protein are removed from the
C-terminus. Whether a particular polypeptide lacking C-terminal
residues of a complete protein retains such immunologic activities
can readily be determined by routine methods described herein and
otherwise known in the art. It is not unlikely that a follistatin-3
mutein with a large number of deleted C-terminal amino acid
residues may retain some biological or immungenic activities. In
fact, peptides composed of as few as six follistatin-3 amino acid
residues may often evoke an immune response.
Accordingly, the present invention further provides polypeptides
having one or more residues deleted from the carboxy terminus of
the amino acid sequence of the follistatin-3 shown in SEQ ID NO:2,
up to the proline residue at position number 6, and polynucleotides
encoding such polypeptides. In particular, the present invention
provides polypeptides comprising the amino acid sequence of
residues 1-m.sup.2 of SEQ ID NO:2, where m.sup.2 is an integer in
the range of 6 to 262, and 6 is the position of the first residue
from the C-terminus of the complete follistatin-3 polypeptide
believed to be required for at least immunogenic activity of the
follistatin-3.
More in particular, the invention provides polynucleotides encoding
polypeptides comprising, or alternatively consisting of, the amino
acid sequence of residues M-1 to F-262; M-1 to N-261; M-1 to E-260;
M-1 to E-259; M-1 to E-258; M-1 to E-257; M-1 to A-256; M-1 to
S-255; M-1 to E-254; M-1 to G-253; M-1 to G-252; M-1 to P-251; M-1
to P-250; M-1 to E-249; M-1 to E-248; M-1 to P-247; M-1 to T-246;
M-1 to G-245; M-1 to A-244; M-1 to C-243; M-1 to S-242; M-l to
G-241; M-1 to A-240; M-1 to H-239; M-1 to R-238; M-1 to V-237; M-1
to G-236; M-1 to 1-235;M-1 to S-234; M-1 to R-233; M-1 to G-232;
M-1 to L-231; M-1 to F-230; M-1 to C-229; M-1 to T-228; M-1 to
A-227; M-1 to Q-226; M-1 to R-225; M-1 to M-224; M-1 to H-223; M-1
to C-222; M-1 to S-221; M-1 to S-220; M-1 to I-219; M-1 to Y-218;
M-1 to T-217; M-1 to V-216; M-1 to N-215; M-1 to N-214; M-1 to
N-213; M-1 to G-212; M-1 to C-211; M-1 to L-210; M-1 to E-209; M-1
to Q-208; M-1 to G-207; M-1 to P-206; M-1 to S-205; M-1 to S-204;
M-1 to P-203; M-1 to V-202; M-1 to-P-201; M-1 to C-200; M-1 to
P-199; M-1 to A-198; M-1 to A-197; M-1 to R-196; M-1 to C-195; M-1
to V-194; M-1 to V-193; M-1 to C-192; M-1 to H-191; M-1 to A-190;
M-1 to S-189; M-1 to G-188; M-1 to T-187; M-1 to Q-186; M-1 to
D-185; M-1 to V-184; M-1 to V-183; M-1 to C-182; M-1 to S-181; M-1
to Q-180; M-1 to P-179; M-1 to R-178; M-1 to P-177; M-1 to C-176;
M-1 to V-175; M-1 to V-174; M-1 to H-173; M-1 to E-172; M-1 to
C-171; M-1 to S-170; M-1 to K-169; M-1 to R-168; M-1 to C-167; M-1
to R-166; M-1 to G-165; M-1 to R-164; M-1 to Y-163; M-1 to M-162;
M-1 to V-161; M-1 to S-160; M-1 to L-159; M-1 to D-158; M-1 to
P-157; M-1 to H-156; M-1 to G-155; M-1 to R-154; M-1 to C-153; M-1
to R-152; M-1 to A-151; M-1 to A-150; M-1 to R-149; M-1 to L-148;
M-1 to E-147; M-1 to C-146; M-1 to E-145; M-1 to D-144; M-1 to
R-143; M-1 to Y-142; M-1 to T-141; M-1 to A-140; M-1 to G-139; M-1
to D-138; M-1 to S-137; M-1 to G-136; M-1 to C-135; M-1 to V-134;
M-1 to Q-133; M-1 to L-132; M-1 to R-131; M-1 to A-130; M-1 to
P-129; M-1 to L-128; M-1 to G-127; M-1 to S-126; M-1 to C-125; M-1
to D-124; M-1 to P-123; M-1 to A-122; M-1 to C-121; M-1 to E-120;
M-1 to C-i 19; M-1 to R-118; M-1 to P-117; M-1 to R-116; M-1 to
G-115; M-1 to G-114; M-1 to L-113; M-1 to M-112; M-1 to R-111; M-1
to C-110; M-1 to A-109; M-1 to K-108; M-1 to G-107; M-1 to P-106;
M-1 to G-105; M-1 to C-104; M-1 to E-103; M-1 to V-102; M-1 to
G-101; M-1 to D-100; M-1 to C-99; M-1 to S-98; M-1 to D-97; M-1 to
K-96; M-1 to C-95; M-1 to P-94; M-1 to L-93; M-1 to C-92; M-1 to
H-91; M-1 to V-90; M-1 to L-89; M-1 to G-88; M-1 to L-87; M-1 to
F-86; M-1 to G-85; M-1 to L-84; M-1 to L-83; M-1 to N-82; M-1 to
I-81; M-1 to K-80; M-1 to N-79; M-1 to G-78; M-1 to P-77; M-1 to
H-76; M-1 to T-75; M-1 to L-74; M-1 to N-73; M-1 to S-72; M-1 to
W-71; M-1 to A-70; M-1 to T-69; M-1 to D-68; M-1 to I-67; M-1 to
N-66; M-1 to G-65; M-1 to S-64; M-1 to A-63; M-1 to C-62; M-1 to
C-61; M-1 to E-60; M-1 to A-59; M-1 to R-58; M-1 to T-57; M-1 to
V-56; M-1 to D-55; M-1 to T-54; M-1 to Q-53; M-1 to L-52; M-1 to
V-51; M-1 to L-50; M-1 to S-49; M-1 to C-48; M-1 to T-47; M-1 to
A-46; M-1 to E-45; M-1 to Q-44; M-1 to G-43; M-1 to Q-42; M-1 to
Q-41; M-1 to L-40; M-1 to W-39; M-1 to C-38; M-1 to V-37; M-1 to
G-36; M-1 to G-35; M-1 to P-34; M-1 to A-33; M-1 to P-32; M-1 to
N-31; M-1 to G-30; M-1 to S-29; M-1 to G-28; M-1 to M-27; M-1 to
S-26; M-1 to S-25; M-1 to V-24; M-1 to F-23; M-1 to G-22; M-1 to
V-21; M-1 to A-20; M-1 to W-19; M-1 to A-18; M-1 to L-17; M-1 to
A-16; M-1 to G-15; M-l to W-14; M-1 to P-13; M-1 to L-12; M-1 to
P-11; M-1 to W-10; M-1 to L-9; M-1 to P-8; M-1 to G-7; M-1 to P-6
of the sequence of the follistatin-3 sequence shown in FIGS. 1A,
1B, and 1C (which is identical to the sequence shown as SEQ ID
NO:2, with the exception that the amino acid residues in FIGS. 1A,
1B, and 1C are numbered consecutively from 1 through 263 from the
N-terminus to the C-terminus, while the amino acid residues in SEQ
ID NO:2 are numbered consecutively from -26 through 237 to reflect
the position of the predicted signal peptide). Polynucleotides
encoding these polypeptides also are provided.
The invention also provides polypeptides having one or more amino
acids deleted from both the amino and the carboxyl termini of a
follistatin-3 polypeptide, which may be described generally as
having residues n.sup.2 -m.sup.2 of FIGS. 1A, 1B, and 1C (SEQ ID
NO:2), where n.sup.2 and m.sup.2 are integers as described
above.
In addition to terminal deletion forms of the protein discussed
above, it also will be recognized by one of ordinary skill in the
art that some amino acid sequences of the follistatin-3 polypeptide
can be varied without significant effect of the structure or
function of the protein. If such differences in sequence are
contemplated, it should be remembered that there will be critical
areas on the protein which determine activity.
Thus, the invention further includes variations of the
follistatin-3 polypeptide which show substantial follistatin-3
polypeptide activity or which include regions of follistatin-3 such
as the protein portions discussed below. Such mutants include
deletions, insertions, inversions, repeats, and type substitutions
selected according to general rules known in the art so as have
little effect on activity. For example, guidance concerning how to
make phenotypically silent amino acid substitutions is provided
wherein the authors indicate that there are two main approaches for
studying the tolerance of an amino acid sequence to change (Bowie,
J. U., et al., Science 247:1306-1310 (1990)),. The first method
relies on the process of evolution, in which mutations are either
accepted or rejected by natural selection. The second approach uses
genetic engineering to introduce amino acid changes at specific
positions of a cloned gene and selections or screens to identify
sequences that maintain functionality.
As the authors state, these studies have revealed that proteins are
surprisingly tolerant of amino acid substitutions. The authors
further indicate which amino acid changes are likely to be
permissive at a certain position of the protein. For example, most
buried amino acid residues require nonpolar side chains, whereas
few features of surface side chains are generally conserved. Other
such phenotypically silent substitutions are described by Bowie and
coworkers (supra) and the references cited therein. Typically seen
as conservative substitutions are the replacements, one for
another, among the aliphatic amino acids Ala, Val, Leu and Ile;
interchange of the hydroxyl residues Ser and Thr, exchange of the
acidic residues Asp and Glu, substitution between the amide
residues Asn and Gln, exchange of the basic residues Lys and Arg
and replacements among the aromatic residues Phe, Tyr.
Thus, the fragment, derivative or analog of the polypeptide of SEQ
ID NO:2, or that encoded by the deposited cDNA, may be (i) one in
which one or more of the amino acid residues are substituted with a
conserved or non-conserved amino acid residue (preferably a
conserved amino acid residue) and such substituted amino acid
residue may or may not be one encoded by the genetic code, or (ii)
one in which one or more of the amino acid residues includes a
substituent group, or (iii) one in which the mature polypeptide is
fused with another compound, such as a compound to increase the
half-life of the polypeptide (for example, polyethylene glycol), or
(iv) one in which the additional amino acids are fused to the above
form of the polypeptide, such as an IgG Fc fusion region peptide or
leader or secretory sequence or a sequence which is employed for
purification of the above form of the polypeptide or a proprotein
sequence. Such fragments, derivatives and analogs are deemed to be
within the scope of those skilled in the art from the teachings
herein.
Thus, the follistatin-3 of the present invention may include one or
more amino acid substitutions, deletions or additions, either from
natural mutations or human manipulation. As indicated, changes are
preferably of a minor nature, such as conservative amino acid
substitutions that do not significantly affect the folding or
activity of the protein (see Table II).
TABLE II Conservative Amino Acid Substitutions. Aromatic
Phenylalanine Tryptophan Tyrosine Hydrophobic Leucine Isoleucine
Valine Polar Glutamine Asparagine Basic Arginine Lysine Histidine
Acidic Aspartic Acid Glutamic Acid Small Alanine Serine Threonine
Methionine Glycine
Embodiments of the invention are directed to polypeptides which
comprise the amino acid sequence of a follistatin-3 polypeptide
described herein, but having an amino acid sequence which contains
at least one conservative amino acid substitution, but not more
than 50 conservative amino acid substitutions, even more
preferably, not more than 40 conservative amino acid substitutions,
still more preferably, not more than 30 conservative amino acid
substitutions, and still even more preferably, not more than 20
conservative amino acid substitutions, when compared with the
follistatin-3 polynucleotide sequence described herein. Of course,
in order of ever-increasing preference, it is highly preferable for
a peptide or polypeptide to have an amino acid sequence which
comprises the amino acid sequence of a follistatin-3 polypeptide,
which contains at least one, but not more than 10, 9, 8, 7, 6, 5,
4, 3, 2 or 1 conservative amino acid substitutions.
In further specific embodiments, the number of substitutions,
additions or deletions in the amino acid sequence of FIGS. 1A, 1B,
and 1C (SEQ ID NO:2), a polypeptide sequence encoded by the
deposited clones, and/or any of the polypeptide fragments described
herein is 75, 70, 60, 50, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6,
5, 4, 3, 2, 1 or 150-50, 100-50, 50-20, 30-20, 20-15, 20-10, 15-10,
10-1, 5-10, 1-5, 1-3 or 1-2.
To improve or alter the characteristics of follistatin-3
polypeptides, protein engineering may be employed. Recombinant DNA
technology known to those skilled in the art can be used to create
novel mutant proteins or muteins including single or multiple amino
acid substitutions, deletions, additions or fusion proteins. Such
modified polypeptides can show, e.g., enhanced activity or
increased stability. In addition, they may be purified in higher
yields and show better solubility than the corresponding natural
polypeptide, at least under certain purification and storage
conditions.
Thus, the invention also encompasses follistatin-3 derivatives and
analogs that have one or more amino acid residues deleted, added,
or substituted to generate follistatin-3 polypeptides that are
better suited for expression, scale up, etc., in the host cells
chosen. For example, cysteine residues can be deleted or
substituted with another amino acid residue in order to eliminate
disulfide bridges; N-linked glycosylation sites can be altered or
eliminated to acheive, for example, expression of a homogeneous
product that is more easily recovered and purified from yeast hosts
which are known to hyperglycosylate N-linked sites. To this end, a
variety of amino acid substitutions at one or both of the first or
third amino acid positions on any one or more of the glycosylation
recognitions sequences in the follistatin-3 polypeptides of the
invention, and/or an amino acid deletion at the second position of
any one or more such recognition sequences will prevent
glycosylation of the follistatin-3 polypeptide at the modified
tripeptide sequence (see, e.g., Miyajima, A., et al., EMBO J. 5(6):
1193-1197 (1986)).
Amino acids in-the follistatin-3 polypeptides of the present
invention that are essential for function can be identified by
methods known in the art, such as site-directed mutagenesis or
alanine-scanning mutagenesis (Cunningham and Wells, Science
244:1081-1085 (1989)). The latter procedure introduces single
alanine mutations at every residue in the molecule. The resulting
mutant molecules are then tested for biological activity such as
receptor binding or in vitro proliferative activity.
Of special interest are substitutions of charged amino acids with
other charged or neutral amino acids which may produce proteins
with highly desirable improved characteristics, such as less
aggregation. Aggregation may not only reduce activity but also be
problematic when preparing pharmaceutical formulations, because
aggregates can be immunogenic (Pinckard, et al., Clin. Exp.
Immunol. 2:331-340 (1967); Robbins, et al., Diabetes 36:838-845
(1987); Cleland, et al., Crit. Rev. Therapeutic Drug Carrier
Systems 10:307-377. (1993)).
A mutational analysis of the two N-linked glycosylation sites
(Asn-95 and Asn-259) of follistatin-1 was conducted by Inouye and
colleagues (Biochem. Biophys. Res. Comm. 179:352-358 (1991)). As
described in the analysis, disruption of either or both of the
N-linked glycosylation sites (by mutation of Thr-97 and Thr-261 to
alanine) had no discernable effect on activin-binding and FSH
secretion. However, results of the same study suggest that
insertion of two amino acid residues (lysine and leucine) between
residues Asn-2 and Cys-3 of follistatin-1 completely abolishes its
inhibitory activity on FSH secretion from the pituitary, as well as
its ability to bind activin. The asparagine and surrounding
residues described in this analysis are weakly conserved between
follistatin-1 and follistatin-3. There are however, two potential
N-linked glycosylation sites in the sequence of follistatin-3 (N-73
and N-215; see FIG. 1A). In addition, 4 out of 5 amino acids making
up the sequence near the amino terminus, at which point Inouye and
coworkers made their two amino acid insertion (supra), are
conserved. Consequently, the extreme amino terminal region of the
predicted mature follistatin-3 polypeptide may have a high
potential for exhibiting a deleterious effect through mutation.
The polypeptides of the present invention are preferably provided
in an isolated form, and preferably are substantially purified. A
recombinantly produced version of the follistatin-3 polypeptide can
be substantially purified by the one-step method described by Smith
and Johnson (Gene 67:31-40 (1988)). Polypeptides of the invention
also can be purified from natural or recombinant sources using
anti-Follistatin-3 antibodies of the invention in methods which are
well known in the art of protein purification.
The invention further provides an isolated follistatin-3
polypeptide comprising an amino acid sequence selected from the
group consisting of: (a) the amino acid sequence of the full-length
follistatin-3 polypeptide having the complete amino acid sequence
shown in SEQ ID NO:2 (i.e., positions -26 to 237 of SEQ ID NO:2);
(b) the amino acid sequence of the full-length follistatin-3
polypeptide having the complete amino acid sequence shown in SEQ ID
NO:2 excepting the N-terminal methionine (i.e., positions -25 to
237 of SEQ ID NO:2); (c) the amino acid sequence of the predicted
mature follistatin-3 polypeptide having the amino acid sequence at
positions 1 to 237 in SEQ ID NO:2; (d) the amino acid sequence of
the full-length follistatin-3 polypeptide having the complete amino
acid sequence encoded by the cDNA clone contained in ATCC.RTM. 4
Deposit No. 209197; (e) the amino acid sequence of the full-length
follistatin-3 polypeptide having the complete amino acid sequence
excepting the N-terminal methionine encoded by the cDNA clone
contained in ATCC.RTM. Deposit No. 209197; and (t) the amino acid
sequence of the mature follistatin-3 polypeptide having the amino
acid sequence encoded by the cDNA clone contained in ATCC.RTM.
Deposit No. 209197. The polypeptides of the present invention also
include polypeptides having an amino acid sequence at least 80%
identical, more preferably at least 90% identical, arid still more
preferably 95%, 96%, 97%, 98% or 99% identical to those described
in (a), (b), (c), (d), (e) or (f) above, as well as polypeptides
having an amino acid sequence with at least 90% similarity, and
more preferably at least 95% similarity, to chose above.
Further polypeptides of the present invention include polypeptides
which have at least 90% similarity, more preferably at least 95%
similarity, and still more preferably at least 96%, 97%, 98% or 99%
similarity to those described above. The polypeptides of the
invention also comprise those which are at least 80% identical,
more preferably at least 90% or 95% identical, still more
preferably at least 96%, 97%, 98% or 99% identical to the
polypeptide encoded by the deposited cDNA or to the polypeptide of
SEQ ID NO:2, and also include portions of such polypeptides with at
least 30 amino acids and more preferably at least 50 amino
acids.
By "% similarity" for two polypeptides is intended a similarity
score produced by comparing the amino acid sequences of the two
polypeptides using the Bestfit program (Wisconsin Sequence Analysis
Package, Version 8 for Unix, Genetics Computer Group, University
Research Park, 575 Science Drive, Madison, Wis. 53711) and the
default settings for determining similarity. Bestfit uses the local
homology algorithm of Smith and Waterman (Advances in Applied
Mathematics 2:482-489, 1981) to find the best segment of similarity
between two sequences.
By a polypeptide having an amino acid sequence at least, for
example, 95% "identical" to a reference amino acid sequence of a
follistatin-3 polypeptide is intended that the amino acid sequence
of the polypeptide is identical to the reference sequence except
that the polypeptide sequence may include up to five amino acid
alterations per each 100 amino acids of the reference amino acid of
the follistatin-3 polypeptide. In other words, to obtain a
polypeptide having an amino acid sequence at least 95% identical to
a reference amino acid sequence, up to 5% of the amino acid
residues in the reference sequence may be deleted or substituted
with another amino acid, or a number of amino acids up to 5% of the
total amino acid residues in the reference sequence may be inserted
into the reference sequence. These alterations of the reference
sequence may occur at the amino or carboxy terminal positions of
the reference amino acid sequence or anywhere between those
terminal positions, interspersed either individually among residues
in the reference sequence or in one or more contiguous groups
within the reference sequence.
As a practical matter, whether any particular polypeptide is at
least 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance,
the amino acid sequence shown in FIGS. 1A, 1B, and 1C (SEQ ID
NO:2), the amino acid sequence encoded by deposited cDNA clone
HDTAH85, or fragments thereof, can be determined conventionally
using known computer programs such the Bestfit program (Wisconsin
Sequence Analysis Package, Version 8 for Unix, Genetics Computer
Group, University Research Park, 575 Science Drive, Madison, Wis.
53711). When using Bestfit or any other sequence alignment program
to determine whether a particular sequence is, for instance, 95%
identical to a reference sequence according to the present
invention, the parameters are set, of course, such that the
percentage of identity is calculated over the full length of the
reference amino acid sequence and that gaps in homology of up to 5%
of the total number of amino acid residues in the reference
sequence are allowed.
In a specific embodiment, the identity between a reference (query)
sequence (a sequence of the present invention) and a subject
sequence, also referred to as a global sequence alignment, is
determined using the FASTDB computer program based on the algorithm
of Brutlag et al. (Comp. App. Biosci. 6:237-245 (1990)). Preferred
parameters used in a FASTDB amino acid alignment are: Matrix=PAM 0,
k-tuple=2, Mismatch Penalty=1, Joining Penalty=20, Randomization
Group Length=0, Cutoff Score=1, Window Size=sequence length, Gap
Penalty=5, Gap Size Penalty=0.05, Window Size=500 or the length of
the subject amino acid sequence, whichever is shorter. According to
this embodiment, if the subject sequence is shorter than the query
sequence due to N- or C-terminal deletions, not because of internal
deletions, a manual correction is made to the results to take into
consideration the fact that the FASTDB program does not account for
N- and C-terminal truncations of the subject sequence when
calculating global percent identity. For subject sequences
truncated at the N- and C-termini, relative to the query sequence,
the percent identity is corrected by calculating the number of
residues of the query sequence that are N- and C-terminal of the
subject sequence, which are not matched/aligned with a
corresponding subject residue, as a percent of the total bases of
the query sequence. A determination of whether a residue is
matched/aligned is determined by results of the FASTDB sequence
alignment. This percentage is then subtracted from the percent
identity, calculated by the above FASTDB program using the
specified parameters, to arrive at a final percent identity score.
This final percent identity score is what is used for the purposes
of this embodiment. Only residues to the N- and C-termini of the
subject sequence, which are not matched/aligned with the query
sequence, are considered for the purposes of manually adjusting the
percent identity score. That is, only query residue positions
outside the farthest N- and C-terminal residues of the subject
sequence. For example, a 90 amino acid residue subject sequence is
aligned with a 100 residue query sequence to determine percent
identity. The deletion occurs at the N-terminus of the subject
sequence and therefore, the FASTDB alignment does not show a
matching/alignment of the first 10 residues at the N-terminus. The
10 unpaired residues represent 10% of the sequence (number of
residues at the N- and C-termini not matched/total number of
residues in the query sequence) so 10% is subtracted from the
percent identity score calculated by the FASTDB program. If the
remaining 90 residues were perfectly matched the final percent
identity would be 90%. In another example, a 90 residue subject
sequence is compared with a 100 residue query sequence. This time
the deletions are internal deletions so there are no residues at
the N- or C-termini of the subject sequence which are not
matched/aligned with the query. In this case the percent identity
calculated by FASTDB is not manually corrected. Once again, only
residue positions outside the N- and C-terminal ends of the subject
sequence, as displayed in the FASTDB alignment, which are not
matched/aligned with the query sequence are manually corrected for.
No other manual corrections are made for the purposes of this
embodiment.
The invention also encompasses fusion proteins in which the
full-length follistatin-3 polypeptide or fragment, variant,
derivative, or analog thereof is fused to an unrelated protein.
These fusion proteins can be routinely designed on the basis of the
follistatin-3 nucleotide and polypeptide sequences disclosed
herein. For example, as one of skill in the art will appreciate,
follistatin-3 polypeptides and fragments (including epitope-bearing
fragments) thereof described herein can be combined with parts of
the constant domain of immunoglobulins (IgG), resulting in chimeric
(fusion) polypeptides. These fusion proteins facilitate
purification and show an increased half-life in vivo. This has been
shown, e.g., for chimeric proteins consisting of the first two
domains of the human CD4-polypeptide and various domains of the
constant regions of the heavy or light chains of mammalian
immunoglobulins (EP A 394,827; Traunecker, et al., Nature 331:84-86
(1988)). Fusion proteins that have a disulfide-linked dimeric
structure due to the IgG part can also be more efficient in binding
and neutralizing other molecules than the monomeric follistatin-3
polypeptide or polypeptide fragments alone (Fountoulakis, et al.,
J. Biochem. 270:3958-3964 (1995)). Examples of follistatin-3 fusion
proteins that are encompassed by the invention include, but are not
limited to, fusion of the follistatin-3 polypeptide sequences to
any amino acid sequence that allows the fusion proteins to be
displayed on the cell surface (e.g. the IgG Fc domain); or fusions
to an enzyme, fluorescent protein, or luminescent protein which
provides a marker function.
The polypeptides of the present invention have uses which include,
but are not limited to, a molecular weight marker on SDS-PAGE gels
or on molecular sieve gel filtration columns using methods well
known to those of skill in the art. Additionally, as described in
detail herein, the polypeptides of the present invention can also
be used to raise polyclonal and monoclonal antibodies, which are
useful in assays for detecting follistatin-3 expression as
described below or as agonists and antagonists capable of enhancing
or inhibiting follistatin-3 function. Further, such polypeptides
can be used in the yeast two-hybrid system to "capture"
follistatin-3 polypeptide binding proteins which are also candidate
agonists and antagonists according to the present invention. The
yeast two hybrid system is described by Fields and Song (Nature
340:245-246 (1989)).
Antibodies
Follistatin-3 polypeptide-specific antibodies for use in the
present invention can be raised against the intact follistatin-3
polypeptide or an antigenic polypeptide fragment thereof, which may
be presented together with a carrier protein, such as an albumin,
to an animal system (such as rabbit or mouse) or, if it is long
enough (at least about 25 amino acids), without a carrier.
As used herein, the term "antibody" (Ab) or "monoclonal antibody"
(Mab) is meant to include intact molecules as well as antibody
fragments (such as, for example, Fab and F(ab')2 fragments) which
are capable of specifically binding to follistatin-3. Fab and
F(ab')2 fragments lack the Fc fragment of intact antibody, clear
more rapidly from the circulation, and may have less non-specific
tissue binding of an intact antibody (Wahl, et al., J. Nucl. Med.
24:316-325 (1983)). Thus, these fragments are preferred.
The antibodies of the present invention may be prepared by any of a
variety of methods. For example, cells expressing the follistatin-3
or an antigenic fragment thereof can be administered to an animal
in order to induce the production of sera containing polyclonal
antibodies. In a preferred method, a preparation of follistatin-3
polypeptide is prepared and purified to render it substantially
free of natural contaminants. Such a preparation is then introduced
into an animal in order to produce polyclonal antisera of greater
specific activity.
In the most preferred method, the antibodies of the present
invention are monoclonal antibodies (or follistatin-3 binding
fragments thereof). Such monoclonal antibodies can be prepared
using hybridoma technology (Kohler, et al., Nature 256:495 (1975);
Kohler, et al., Eur. J. Immunol. 6:511 (1976); Kohler, et al., Eur.
J. Immunol. 6:292 (1976); Hammerling, et al., in: Monoclonal
Antibodies and T-Cell Hybridomas, Elsevier, N.Y., (1981) pp.
563-681)). In general, such procedures involve immunizing an animal
(preferably a mouse) with a follistatin-3 antigen or, more
preferably, with a follistatin-3-expressing cell. Suitable cells
can be recognized by their capacity to bind anti-Follistatin-3
antibody. Such cells may be cultured in any suitable tissue culture
medium; however, it is preferable to culture cells in Earle's
modified Eagle's medium supplemented with 10% fetal bovine serum
(inactivated at about 56.degree. C), and supplemented with about 10
.mu.g/ml of nonessential amino acids, about 1,000 U/ml of
penicillin, and about 100 .mu.g/ml of streptomycin. The splenocytes
of such mice are extracted and fused with a suitable myeloma cell
line. Any suitable myeloma cell line may be employed in accordance
with the present invention; however, it is preferable to employ the
parent myeloma cell line (SP2O), available from the American Type
Culture Collection, Rockville, Md. After fusion, the resulting
hybridoma cells are selectively maintained in HAT medium, and then
cloned by limiting dilution as described by Wands and colleagues
(Gastroenterology 80:225-232 (1981)). The hybridoma cells obtained
through such a selection are then assayed to identify clones which
secrete antibodies capable of binding the follistatin-3
antigen.
Alternatively, additional antibodies capable of binding to the
follistatin-3 antigen may be produced in a two-step procedure
through the use of anti-idiotypic antibodies. Such a method makes
use of the fact that antibodies are themselves antigens, and that,
therefore, it is possible to obtain an antibody which binds to a
second antibody. In accordance with this method,
follistatin-3-specific antibodies are used to immunize an animal,
preferably a mouse. The splenocytes of such an animal are then used
to produce hybridoma cells, and the hybridoma cells are screened to
identify clones which produce an antibody whose ability to bind to
the follistatin-3-specific antibody can be blocked by the
follistatin-3 antigen. Such antibodies comprise anti-idiotypic
antibodies to the follistatin-3-specific antibody and can be used
to immunize an animal to induce formation of further
follistatin-3-specific antibodies.
It will be appreciated that Fab and F(ab')2 and other fragments of
the antibodies of the present invention may be used according to
the methods disclosed herein. Such fragments are typically produced
by proteolytic cleavage, using enzymes such as papain (to produce
Fab fragments) or pepsin (to produce F(ab')2 fragments).
Alternatively, follistatin-3-binding fragments can be produced
through the application of recombinant DNA technology or through
synthetic chemistry.
For in vivo use of anti-Follistatin-3 in humans, it may be
preferable to use "humanized" chimeric monoclonal antibodies. Such
antibodies can be produced using genetic constructs derived from
hybridoma cells producing the monoclonal antibodies described
above. Methods for producing chimeric antibodies are known in the
art (Morrison, Science 229:1202 (1985); Oi, et al., BioTechniques
4:214 (1986); Cabilly, et al., U.S. Pat. No. 4,816,567; Taniguchi,
et al., EP 171496; Morrison, et al., EP 173494; Neuberger, et al.,
WO 8601533; Robinson, et al., WO 8702671; Boulianne, et al., Nature
312:643 (1984); Neuberger, et al., Nature 314:268 (1985).
Reproductive System--and Cell Growth and Differentiation-Related
Disorders
Diagnosis
The present inventors have discovered that follistatin-3 is
expressed not only in Hodgkin's Lymphoma, but also in synovial
fibroblasts, gall bladder, resting and serum-induced smooth muscle,
testes, Merkel cells, HEL cells, hippocampus, TNF-.alpha.-and
IFN-induced epithelial cells, keratinocyte, amygdala depression,
HL-60 cells, hepatoma, progesterone-treated epidermal cells,
endothelial cells, HSC 172 cells, epithelioid sarcoma, activated
T-cells, breast lymph node, pancreatic carcinoma, fetal dura mater,
fetal lung, epididymis, placenta, dendritic cells, rejected kidney,
and uterine cancer. For a number of reproductive system-related
disorders and disorders related to the regulation of cell growth
and differentiation, substantially altered (increased or decreased)
levels of follistatin-3 gene expression can be detected in
reproductive system tissue or other cells or bodily fluids (e.g.,
sera, plasma, urine, synovial fluid or spinal fluid) taken from an
individual having such a disorder, relative to a "standard"
follistatin-3 gene expression level, that is, the follistatin-3
expression level in reproductive system tissues or bodily fluids
from an individual not having the reproductive system or cell
growth and differentiation disorder. Thus, the invention provides a
diagnostic method useful during diagnosis of a reproductive system
or cell growth and differentiation disorder, which involves
measuring the expression level of the gene encoding the
follistatin-3 polypeptide in reproductive system tissue or other
cells or body fluid from an individual and comparing the measured
gene expression level with a standard-follistatin-3 gene expression
level, whereby an increase or decrease in the gene expression level
compared to the standard is indicative of a reproductive or cell
growth and differentiation system disorder.
In particular, it is believed that certain tissues in mammals with
cancer of various cells and tissues of the reproductive or other
systems express significantly reduced levels of the follistatin-3
polypeptide and mRNA encoding the follistatin-3 polypeptide when
compared to a corresponding "standard" level. Further, it is
believed that enhanced levels of the follistatin-3 polypeptide can
be detected in certain body fluids (e.g., sera, plasma, urine, and
spinal fluid) from mammals with such a cancer when compared to sera
from mammals of the same species not having the cancer.
Thus, the invention provides a diagnostic method useful during
diagnosis of reproductive system or cell growth and differentiation
disorders, including cancers of these systems, which involves
measuring the expression level of the gene encoding the
follistatin-3 polypeptide in reproductive system tissue or other
cells or body fluid from an individual and comparing the measured
gene expression level with a standard follistatin-3 gene expression
level, whereby an increase or decrease in the gene expression level
compared to the standard is indicative of a reproductive system
disorder or a disorder of the regulation of cell growth and
differentiation.
Where a diagnosis of a disorder in the reproductive or other system
including diagnosis of a tumor, has already been made according to
conventional methods, the present invention is useful as a
prognostic indicator, whereby patients exhibiting depressed
follistatin-3 gene expression will experience a worse clinical
outcome relative to patients expressing the gene at a level nearer
the standard level.
By "assaying the expression level of the gene encoding the
follistatin-3 polypeptide" is intended qualitatively or
quantitatively measuring or estimating the level of the
follistatin-3 polypeptide or the level of the mRNA encoding the
follistatin-3 polypeptide in a first biological sample either
directly (e.g., by determining or estimating absolute polypeptide
level or mRNA level) or relatively (e.g., by comparing to the
follistatin-3 polypeptide level or mRNA level in a second
biological sample). Preferably, the follistatin-3 polypeptide level
or mRNA level in the first biological sample is measured or
estimated and compared to a standard follistatin-3 polypeptide
level or mRNA level, the standard being taken from a second
biological sample obtained from an individual not having the
disorder or being determined by averaging levels from a population
of individuals not having a disorder of the reproductive system or
of regulation of cell growth and differentiation. As will be
appreciated in the art, once a standard follistatin-3 polypeptide
level or mRNA level is known, it can be used repeatedly as a
standard for comparison.
By "biological sample" is intended any biological sample obtained
from an individual, body fluid, cell line, tissue culture, or other
source which contains follistatin-3 polypeptide or mRNA. As
indicated, biological samples include body fluids (such as sera,
plasma, urine, synovial fluid and spinal fluid) which contain free
follistatin-3 polypeptide, reproductive system tissue, and other
tissue sources found to express complete or mature follistatin-3 or
a follistatin-3 receptor. Methods for obtaining tissue biopsies and
body fluids from mammals are well known in the art. Where the
biological sample is to include mRNA, a tissue biopsy is the
preferred source.
The present invention is useful for diagnosis or treatment of
various reproductive system-related disorders and disorders of the
regulation of cell growth and differentiation in mammals,
preferably humans. Such disorders include tumors, cancers,
interstitial lung disease, and any disregulation of the growth and
differentiation patterns of cell function including, but not
limited to, autoimmunity, arthritis, leukemias, lymphomas,
immunosuppression, immunity, humoral immunity, inflammatory bowel
disease, myelosuppression and the like.
Total cellular RNA can be isolated from a biological sample using
any suitable technique such as the single-step
guanidinium-thiocyanate-phenol-chloroform method described by
Chomczynski and Sacchi (Anal. Biochem. 162:156-159 (1987)). Levels
of mRNA encoding the follistatin-3 polypeptide are then assayed
using any appropriate method. These include Northern blot analysis,
S1 nuclease mapping, the polymerase chain reaction (PCR), reverse
transcription in combination with the polymerase chain reaction
(RT-PCR), and reverse transcription in combination with the ligase
chain reaction (RT-LCR).
Assaying follistatin-3 polypeptide levels in a biological sample
can occur using antibody-based techniques. For example,
follistatin-3 polypeptide expression in tissues can be studied with
classical immunohistological methods (Jalkanen, M., et al., J.
Cell. Biol. 101:976-985 (1985); Jalkanen, M., et al., J. Cell.
Biol. 105:3087-3096 (1987)). Other antibody-based methods useful
for detecting follistatin-3 polypeptide gene expression include
immunoassays, such as the enzyme linked immunosorbent assay (ELISA)
and the radioimmunoassay (RIA). Suitable antibody assay labels are
known in the art and include enzyme labels, such as, glucose
oxidase, and radioisotopes, such as iodine (.sup.125 I, .sup.121
I), carbon (.sup.14 C), sulfur (.sup.35 S), tritium (.sup.3 H),
indium (.sup.112 In), and technetium (.sup.99m Tc), and fluorescent
labels, such as fluorescein and rhodamine, and biotin.
In addition to assaying follistatin-3 polypeptide levels in a
biological sample obtained from an individual, follistatin-3
polypeptide can also be detected in vivo by imaging. Antibody
labels or markers for in vivo imaging of follistatin-3 polypeptide
include those detectable by X-radiography, NMR or ESR. For
X-radiography, suitable labels include radioisotopes such as barium
or cesium, which emit detectable radiation but are not overtly
harmful to the subject. Suitable markers for NMR and ESR include
those with a detectable characteristic spin, such as deuterium,
which may be incorporated into the antibody by labeling of
nutrients for the relevant hybridoma.
A follistatin-3 polypeptide-specific antibody or antibody fragment
which has been labeled with an appropriate detectable imaging
moiety, such as a radioisotope (for example, .sup.131 I, .sup.112
In, .sup.99m Tc), a radio-opaque substance, or a material
detectable by nuclear magnetic resonance, is introduced (for
example, parenterally, subcutaneously or intraperitoneally) into
the mammal to be examined for immune system disorder. It will be
understood in the art that the size of the subject and the imaging
system used will determine the quantity of imaging moiety needed to
produce diagnostic images. In the case of a radioisotope moiety,
for a human subject, the quantity of radioactivity injected will
normally range from about 5 to 20 millicuries of .sup.99m Tc. The
labeled antibody or antibody fragment will then preferentially
accumulate at the location of cells which contain follistatin-3
polypeptide. In vivo tumor imaging is described by Burchiel and
coworkers (Chapter 13 in Tumor Imaging: The Radiochemical Detection
of Cancer, Burchiel, S. W. and Rhodes, B. A., eds., Masson
Publishing Inc. (1982)).
Treatment
As noted above, follistatin-3 polynucleotides and polypeptides are
useful for diagnosis of conditions involving abnormally high or low
expression of follistatin-3 activities. Given the cells and tissues
where follistatin-3 is expressed as well as the activities
modulated by follistatin-3, it is readily apparent that a
substantially altered (increased or decreased) level of expression
of follistatin-3 in an individual compared to the standard or
"normal" level produces pathological conditions related to the
bodily system(s) in which follistatin-3 is expressed and/or is
active.
It will also be appreciated by one of ordinary skill that, since
the follistatin-3 polypeptide of the invention is a member of the
inhibin-related protein family the mature secreted form of the
protein may be released in soluble form from the cells which
express the follistatin-3 by proteolytic cleavage. Therefore, when
follistatin-3 mature form is added from an exogenous source to
cells, tissues or the body of an individual, the protein will exert
its physiological activities on its target cells of that
individual.
Therefore, it will be appreciated that conditions caused by a
decrease in the standard or normal level of Follistatin-3 activity
in an individual, particularly disorders of the reproductive
system, can be treated by administration of follistatin-3
polypeptide (in the form of the mature protein). Thus, the
invention also provides a method of treatment of an individual in
need of an increased level of follistatin-3 activity comprising
administering to such an individual a pharmaceutical composition
comprising an amount of an isolated follistatin-3 polypeptide of
the invention, particularly a mature form of the follistatin-3
protein of the invention, effective to increase the follistatin-3
activity level in such an individual.
Follistatin-3 may be used to treat male sterility by its innate
ability to bind activin and, as a result, prevent activin-binding
to its receptor. Activin receptor-binding results in a suppression
of FSH secretion. Increased levels of FSH, in turn, result in an
increase in spermatogenesis (Ying, S.-Y. Endocrine Rev. 9:267-293
(1988)). Thus, a decrease in the effective concentration of activin
will result in an FSH-mediated increase in spermatogenesis. In
addition, since activin elicits a number of biological effects
including the modulation of gonadal androgen biosynthesis (Hsueh,
A. J. W., et al., Proc. Natl. Acad. Sci. USA 84:5082-5086 (1987)),
the attenuation of growth hormone secretion (Bilezikjian, L. M., et
al., Endocrinology 126:2369-2376 (1990), the promotion of erythroid
cell differentiation (Eto, Y., et al., Biochem. Biophys. Res. Comm.
142:1095-1103 (1987)), the induction of mesoderm formation (Smith,
J. C., et al., Nature 345:729-731 (1990)), and the maintenance of
nerve cell survival (Schubert, D., et al., Nature 344:868-870
(1990)), and since follistatin-3 directly inhibits activin
acitivity, follistatin-3 may be used to therapeutically regulate,
as well as diagnostically evaluate, the conditions and events
listed above. Follistatin-3 may also be used to inhibit the
activin-induced differentiation of follicular granulosa cells
(Nakamura, T., et al., Biochim. Biophys. Acta 1135:103-109 (1992)).
Follistatin-3 may be used therapeutically to regulate autocrine
endothelial cell activity and, as a result, induce angiogenesis
(Kozian, D. H., et al., Lab. Invest. 76:267-276 (1997)).
Follistatin-3 may also be used to inhibit the activity of activin
and thereby prevent the observed activin-mediated inhibition of
basal and androgen-stimulated proliferation and induction of
apoptosis (Wang, Q. F., et al., Endocrinology 137:5476-5483
(1996)). Treatment to increase the expression or the presence of
follistatin-3 may be used to inhibit the progression of gonadotroph
adenomas, osteosarcomas, hepatomas, and other tumors and cancers
including bone, breast, colon, lymphomas, leukemias, epithelial
carcinomas, pancreatic, stomach, liver, lung, melanoma, prostate,
ovarian, uterine, bladder, gliomas, retinoblastomas, sarcomas, and
the like (Penabad, J. L., et al., J. Clin. Endocrinol. Metab.
81:3397-3403 (1996); Kato, M. V., et al., Oncogene 12:1361-1364
(1996)). Follistatin-3 may also be employed to stimulate wound
healing. In this same manner, follistatin-3 may also be employed to
treat other fibrotic disorders, including liver cirrhosis,
osteoarthritis and pulmonary fibrosis. Follistatin-3 also increases
the presence of eosinophils which have the distinctive function of
killing the larvae of parasites that invade tissues, as in
schistosomiasis, trichinosis and ascariasis. It may also be
employed to regulate hematopoiesis, by regulating the activation
and differentiation of various hematopoietic progenitor cells, for
example, to release mature leukocytes from the bone marrow
following chemotherapy, i.e., in stem cell mobilization.
Follistatin-3 may also be employed to treat sepsis. Follistatin-3
may also be used to treat a number of disease states known to those
of skill in the art which may be therapeutically regulated by
exploiting the prohibitive interation of follistatin-3 with the
activin molecule.
Formulations and Administration
The follistatin-3 polypeptide composition will be formulated and
dosed in a fashion consistent with good medical practice, taking
into account the clinical condition of the individual patient
(especially the side effects of treatment with follistatin-3
polypeptide alone), the site of delivery of the follistatin-3
polypeptide composition, the method of administration, the
scheduling of administration, and other factors known to
practitioners. The "effective amount" of follistatin-3 polypeptide
for purposes herein is thus determined by such considerations.
As a general proposition, the total pharmaceutically effective
amount of follistatin-3 polypeptide administered parenterally per
dose will be in the range of about 1 .mu.g/kg/day to 10 mg/kg/day
of patient body weight, although, as noted above, this will be
subject to therapeutic discretion. More preferably, this dose is at
least 0.01 mg/kg/day, and most preferably for humans between about
0.01 and 1 mg/kg/day for the hormone. If given continuously, the
follistatin-3 polypeptide is typically administered at a dose rate
of about 1 .mu.g/kg/hour to about 50 .mu.g/kg/hour, either by 1-4
injections per day or by continuous subcutaneous infusions, for
example, using a mini-pump. An intravenous bag solution may also be
employed. The length of treatment needed to observe changes and the
interval following treatment for responses to occur appears to vary
depending on the desired effect.
Pharmaceutical compositions containing the follistatin-3 of the
invention may be administered orally, rectally, parenterally,
intracistemally, intravaginally, intraperitoneally, topically (as
by powders, ointments, drops or transdermal patch), bucally, or as
an oral or nasal spray. By "pharmaceutically acceptable carrier" is
meant a non-toxic solid, semisolid or liquid filler, diluent,
encapsulating material or formulation auxiliary of any type. The
term "parenteral" as used herein refers to modes of administration
which include intravenous, intramuscular, intraperitoneal,
intrasternal, subcutaneous and intraarticular injection and
infusion.
The follistatin-3 polypeptide is also suitably administered by
sustained-release systems. Suitable examples of sustained-release
compositions include semi-permeable polymer matrices in the form of
shaped articles, e.g., films, or mirocapsules. Sustained-release
matrices include polylactides (U.S. Pat. No. 3,773,919, EP 58,481),
copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (Sidman,
U., et al., Biopolymers 22:547-556 (1983)), poly (2-hydroxyethyl
methacrylate; Langer, R., et al., J. Biomed. Mater. Res. 15:167-277
(1981), and Langer, R., Chem. Tech. 12:98-105 (1982)), ethylene
vinyl acetate (Langer, R., et al., Id.) or
poly-D-(-)-3-hydroxybutyric acid (EP 133,988). Sustained-release
follistatin-3 polypeptide compositions also include liposomally
entrapped follistatin-3 polypeptide. Liposomes containing
follistatin-3 polypeptide are prepared by methods known in the art
(DE 3,218,121; Epstein, et al., Proc. Natl. Acad. Sci. (USA)
82:3688-3692 (1985); Hwang, et al., Proc. Natl. Acad. Sci. (USA)
77:4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046; EP 143,949;
EP 142,641; Japanese Pat. Appl. 83-118008; U.S. Pat. Nos. 4,485,045
and 4,544,545; and EP 102,324). Ordinarily, the liposomes are of
the small (about 200-800 Angstroms) unilamellar type in which the
lipid content is greater than about 30 mol. percent cholesterol,
the selected proportion being adjusted for the optimal
follistatin-3 polypeptide therapy.
For parenteral administration, in one embodiment, the follistatin-3
polypeptide is formulated generally by mixing it at the desired
degree of purity, in a unit dosage injectable form (solution,
suspension, or emulsion), with a pharmaceutically acceptable
carrier, i.e., one that is non-toxic to recipients at the dosages
and concentrations employed and is compatible with other
ingredients of the formulation. For example, the formulation
preferably does not include oxidizing agents and other compounds
that are known to be deleterious to polypeptides.
Generally, the formulations are prepared by contacting the
follistatin-3 polypeptide uniformly and intimately with liquid
carriers or finely divided solid carriers or both. Then, if
necessary, the product is shaped into the desired formulation.
Preferably the carrier is a parenteral carrier, more preferably a
solution that is isotonic with the blood of the recipient. Examples
of such carrier vehicles include water, saline, Ringer's solution,
and dextrose solution. Non-aqueous vehicles such as fixed oils and
ethyl oleate are also useful herein, as well as liposomes.
The carrier suitably contains minor amounts of additives such as
substances that enhance isotonicity and chemical stability. Such
materials are non-toxic to recipients at the dosages and
concentrations employed, and include buffers such as phosphate,
citrate, succinate, acetic acid, and other organic acids or their
salts; antioxidants such as ascorbic acid; low molecular weight
(less than about ten residues) polypeptides, e.g., polyarginine or
tripeptides; proteins, such as serum albumin, gelatin, or
immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;
amino acids, such as glycine, glutamic acid, aspartic acid, or
arginine; monosaccharides, disaccharides, and other carbohydrates
including cellulose or its derivatives, glucose, manose, or
dextrins; chelating agents such as EDTA; sugar alcohols such as
mannitol or sorbitol; counterions such as sodium; and/or nonionic
surfactants such as polysorbates, poloxamers, or PEG.
The follistatin-3 polypeptide is typically formulated in such
vehicles at a concentration of about 0.1 mg/ml to 100 mg/ml,
preferably 1-10 mg/ml, at a pH of about 3 to 8. It will be
understood that the use of certain of the foregoing excipients,
carriers, or stabilizers will result in the formation of
follistatin-3 polypeptide salts.
Follistatin-3 polypeptide to be used for therapeutic administration
must be sterile. Sterility is readily accomplished by filtration
through sterile filtration membranes (e.g., 0.2 micron membranes).
Therapeutic follistatin-3 polypeptide compositions generally are
placed into a container having a sterile access port, for example,
an intravenous solution bag or vial having a stopper pierceable by
a hypodermic injection needle.
Follistatin-3 polypeptide ordinarily will be stored in unit or
multi-dose containers, for example, sealed ampoules or vials, as an
aqueous solution or as a lyophilized formulation for
reconstitution. As an example of a lyophilized formulation, 10-ml
vials are filled with 5 ml of sterile-filtered 1% (w/v) aqueous
follistatin-3 polypeptide solution, and the resulting mixture is
lyophilized. The infusion solution is prepared by reconstituting
the lyophilized follistatin-3 polypeptide using bacteriostatic
water-for-injection (WFI).
The invention also provides a pharmaceutical pack or kit comprising
one or more containers filled with one or more of the ingredients
of the pharmaceutical compositions of the invention. Associated
with such container(s) can be a notice in the form prescribed by a
governmental agency regulating the manufacture, use or sale of
pharmaceuticals or biological products, which notice reflects
approval by the agency of manufacture, use or sale for human
administration. In addition, the polypeptides of the present
invention may be employed in conjunction with other therapeutic
compounds.
Agonists and Antagonists--Assays and Molecules
The invention also provides a method of screening compounds to
identify those which enhance or block the action of follistatin-3
on cells, such as its interaction with follistatin-3-binding
molecules such as activin, an activin-like molecule, or a
follistatin-3 receptor molecule. An agonists is a compound which
increases the natural biological functions of follistatin-3 or
which functions in a manner similar to follistatin-3, while
antagonists decrease or eliminate such functions.
In another aspect of this embodiment the invention provides a
method for identifying an activin-like molecule or a receptor
protein or other ligand-binding protein which binds specifically to
a follistatin-3 polypeptide. For example, a cellular compartment,
such as a membrane or a preparation thereof, may be prepared from a
cell that expresses a molecule that binds follistatin-3. The
preparation is incubated with labeled follistatin-3 and complexes
of follistatin-3 bound to the activin-like molecule, receptor or
other binding protein are isolated and characterized according to
routine methods known in the art. Alternatively, the follistatin-3
polypeptide may be bound to a solid support so that binding
molecules solubilized from cells are bound to the column and then
eluted and characterized according to routine methods.
In the assay of the invention for agonists or antagonists, a
cellular compartment, such as a membrane or a preparation thereof,
may be prepared from a cell that expresses a molecule that binds
follistatin-3, such as a molecule of a signaling or regulatory
pathway modulated by follistatin-3. The preparation is incubated
with labeled follistatin-3 in the absence or the presence of a
candidate molecule which may be a follistatin-3 agonists or
antagonist. The ability of the candidate molecule to bind the
binding molecule is reflected in decreased binding of the labeled
ligand. Molecules which bind gratuitously, i.e., without inducing
the effects of follistatin-3 on binding the follistatin-3 binding
molecule, are most likely to be good antagonists. Molecules that
bind well and elicit effects that are the same as or closely
related to follistatin-3 are agonists.
Follistatin-3-like effects of potential agonists and antagonists
may by measured, for instance, by determining activity of a second
messenger system following interaction of the candidate molecule
with a cell or appropriate cell preparation, and comparing the
effect with that of follistatin-3 or molecules that elicit the same
effects as follistatin-3. Second messenger systems that may be
useful in this regard include but are not limited to AMP guanylate
cyclase, ion channel or phosphoinositide hydrolysis second
messenger systems.
Another example of an assay for follistatin-3 antagonists is a
competitive assay that combines follistatin-3 and a potential
antagonist with membrane-bound follistatin-3 receptor molecules or
recombinant follistatin-3 receptor molecules under appropriate
conditions for a competitive inhibition assay. Follistatin-3 can be
labeled, such as by radioactivity, such that the number of
follistatin-3 molecules bound to a receptor molecule can be
determined accurately to assess the effectiveness of the potential
antagonist.
Potential antagonists include small organic molecules, peptides,
polypeptides and antibodies that bind to a polypeptide of the
invention and thereby inhibit or extinguish its activity. Potential
antagonists also may be small organic molecules, a peptide, a
polypeptide such as a closely related protein or antibody that
binds the same sites on a binding molecule, such as a receptor
molecule, without inducing follistatin-3-induced activities,
thereby preventing the action of follistatin-3 by excluding
follistatin-3 from binding.
Other potential antagonists include antisense molecules. Antisense
technology can be used to control gene expression through antisense
DNA or RNA or through triple-helix formation. Antisense techniques
are discussed in a number of studies (for example, Okano, J.
Neurochem. 56:560 (1991); "Oligodeoxynucleotides as Antisense
Inhibitors of Gene Expression." CRC Press, Boca Raton, Fla.
(1988)). Triple helix formation is discussed in a number of
studies, as well (for instance, Lee, et al., Nucleic Acids Research
6:3073 (1979); Cooney, et al., Science 241:456 (1988); Dervan, et
al., Science 251:1360 (1991)). The methods are based on binding of
a polynucleotide to a complementary DNA or RNA. For example, the 5'
coding portion of a polynucleotide that encodes the mature
polypeptide of the present invention may be used to design an
antisense RNA oligonucleotide of from about 10 to 40 base pairs in
length. A DNA oligonucleotide is designed to be complementary to a
region of the gene involved in transcription thereby preventing
transcription and the production of follistatin-3. The antisense
RNA oligonucleotide hybridizes to the mRNA in vivo and blocks
translation of the mRNA molecule into follistatin-3 polypeptide.
The oligonucleotides described above can also be delivered to cells
such that the antisense RNA or DNA may be expressed in vivo to
inhibit production of follistatin-3.
The agonists and antagonists may be employed in a composition with
a pharmaceutically acceptable carrier, e.g., as described
above.
Antagonists of follistatin-3 may be employed, for instance, to
treat a deficiency in FSH, estrogen, and other hormones.
Follistatin-1 and follistatin-3 are potent inhibitors of FSH and
estrogen production and secretion. As a result, a deficiency of
these or related hormones may be corrected or ameliorated through
the use of a follistatin-3 antagonist. A follistatin-3 antagonist
may be used to prevent or inhibit or reduce the production of
spermatozoa by inhibiting the interaction of follistatin-3 with
activin. Antagonists of follistatin-3 may also be used to modulate
gonadal androgen biosynthesis, attenuate growth hormone secretion,
promote the differentiation of follicular granulosa, erythroid, and
other cell types, induce mesoderm formation, and increase the
survival of nerve cells. A follistatin-3 antagonist may be used to
inhibit angiogenesis related to or independent of tumorigenesis.
Follistatin-3 antagonists may also be useful in increasing the
activity of activin and thereby increasing the observed
activin-mediated inhibition of basal and androgen-stimulated
proliferation and induction of apoptosis. Antagonists of
follistatin-3 may be used to regulate the hormonal and growth
factor environment, and consequently, the activity of macrophages
and their precursors, and of neutrophils, basophils, B lymphocytes
and some T-cell subsets, e.g., activated and CD8 cytotoxic T cells
and natural killer cells, in certain auto-immune and chronic
inflammatory and infective diseases. Examples of auto-immune
diseases include multiple sclerosis, and insulin-dependent
diabetes. The antagonists may also be employed to treat infectious
diseases including silicosis, sarcoidosis, idiopathic pulmonary
fibrosis by alterring the activation state of mononuclear
phagocytes. They may also be employed to treat idiopathic
hyper-eosinophilic syndrome by preventing eosinophil production and
activation. Endotoxic shock may also be treated by the antagonists
by preventing the activation of macrophages. Any of the above
antagonists may be employed in a composition with a
pharmaceutically acceptable carrier, e.g., as hereinafter
described.
Gene Mapping
The nucleic acid molecules of the present invention are also
valuable for chromosome identification. The sequence is
specifically targeted to and can hybridize with a particular
location on an individual human chromosome. Moreover, there is a
current need for identifying particular sites on the chromosome.
Few chromosome marking reagents based on actual sequence data
(repeat polymorphisms) are presently available for marking
chromosomal location. The mapping of DNAs to chromosomes according
to the present invention is an important first step in correlating
those sequences with genes associated with disease.
In certain preferred embodiments in this regard, the cDNA herein
disclosed is used to clone genomic DNA of a follistatin-3 gene.
This can be accomplished using a variety of well known techniques
and libraries, which generally are available commercially. The
genomic DNA then is used for in situ chromosome mapping using well
known techniques for this purpose.
In addition, in some cases, sequences can be mapped to chromosomes
by preparing PCR primers (preferably 15-25 bp) from the cDNA.
Computer analysis of the 3' untranslated region of the gene is used
to rapidly select primers that do not span more than one exon in
the genomic DNA, thus complicating the amplification process. These
primers are then used for PCR screening of somatic cell hybrids
containing individual human chromosomes. Fluorescence in situ
hybridization ("FISH") of a cDNA clone to a metaphase chromosomal
spread can be used to provide a precise chromosomal location in one
step. This technique can be used with probes from the cDNA as short
as 50 or 60 bp (for a review of this technique, see Verma, et al.,
Human Chromosomes: A Manual Of Basic Techniques, Pergamon Press,
New York. (1988)).
Once a sequence has been mapped to a precise chromosomal location,
the physical position of the sequence on the chromosome can be
correlated with genetic map data. Such data are found, for example,
on the World Wide Web (McKusick, V. Mendelian Inheritance In Man,
available on-line through Johns Hopkins University, Welch Medical
Library). The relationship between genes and diseases that have
been mapped to the same chromosomal region are then identified
through linkage analysis (coinheritance of physically adjacent
genes).
Next, it is necessary to determine the differences in the cDNA or
genomic sequence between affected and unaffected individuals. If a
mutation is observed in some or all of the affected individuals but
not in any normal individuals, then the mutation is likely to be
the causative agent of the disease.
Having generally described the invention, the same will be more
readily understood by reference to the following examples, which
are provided by way of illustration and are not intended as
limiting.
EXAMPLES
Example 1(a)
Expression and Purification of "His-tagged" Follistatin-3 in E.
coli
The bacterial expression vector pHE-4 is used for bacterial
expression in this example. pHE-4 encodes ampicillin antibiotic
resistance ("Ampr") and contains a bacterial origin of replication
("ori"), an IPTG inducible promoter, a ribosome binding site
("RBS"), six codons encoding histidine residues that allow affinity
purification using nickel-nitrilo-tri-acetic acid ("Ni-NTA")
affinity resin sold by QIAGEN, Inc., supra, and suitable single
restriction enzyme cleavage sites. These elements are arranged such
that an inserted DNA fragment encoding a polypeptide expresses that
polypeptide with the six His residues (i.e., a "6.times. His tag")
covalently linked to the amino terminus of that polypeptide.
The DNA sequence encoding the desired portion of follistatin-3
comprising the mature form of the follistatin-3 amino acid sequence
is amplified from the deposited cDNA clone using PCR
oligonucleotide primers which anneal to the amino terminal
sequences of the desired portion of follistatin-3 and to sequences
in the deposited construct 3' to the cDNA coding sequence.
Additional nucleotides containing restriction sites to facilitate
cloning in the pHE-4 vector are added to the 5' and 3' primer
sequences, respectively.
For cloning the mature form of the follistatin-3 protein, the 5'
primer has the sequence 5' TCA CGC CAT ATG GGC TCG GGG AAC C 3'
(SEQ ID NO:12) containing the underlined Nde I restriction site
followed by 16 nucleotides of the amino terminal coding sequence of
the mature follistatin-3 sequence in SEQ ID NO:2. One of ordinary
skill in the art would appreciate, of course, that the point in the
protein coding sequence where the 5' primer begins may be varied to
amplify a DNA segment encoding any desired portion of the complete
follistatin-3 protein shorter or longer than the mature form of the
protein. The 3' primer has the sequence 5' CAT CCG GGT ACC TTA TTA
CAC GAA GTT CTC TTC CTC TTC TG 3' (SEQ ID NO:13) containing the
underlined Asp 718 restriction site followed by two stop codons and
23 nucleotides complementary to the 3' end of the coding sequence
of the follistatin-3 DNA sequence in FIGS. 1A, 1B, and 1C.
The amplified follistatin-3 DNA fragment and the vector pHE4 are
digested with Nde I and Asp 718 and the digested DNAs are then
ligated together. Insertion of the follistatin-3 DNA into the
restricted pHE4 vector places the follistatin-3 protein coding
region downstream from the IPTG-inducible promoter and in-frame
with an initiating AUG and the six histidine codons.
The ligation mixture is transformed into competent E. coli cells
using standard procedures such as those described by Sambrook and
colleagues (Molecular Cloning: a Laboratory Manual, 2nd Ed.; Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)).
E. coli strain M15/rep4, containing multiple copies of the plasmid
pREP4, which expresses the lac repressor and confers kanamycin
resistance ("Kanr"), is used in carrying out the illustrative
example described herein. This strain, which is only one of many
that are suitable for expressing follistatin-3 protein, is
available commercially (QIAGEN, Inc., supra). Transformants are
identified by their ability to grow on LB plates in the presence of
ampicillin and kanamycin. Plasmid DNA is isolated from resistant
colonies and the identity of the cloned DNA confirmed by
restriction analysis, PCR and DNA sequencing.
Clones containing the desired constructs are grown overnight
("O/N") in liquid culture in LB media supplemented with both
ampicillin (100 .mu.g/ml) and kanamycin (25 .mu.g/ml). The O/N
culture is used to inoculate a large culture, at a dilution of
approximately 1:25 to-1:250. The cells are grown to an optical
density at 600 nm ("OD600") of between 0.4 and 0.6.
Isopropyl-.beta.-D-thiogalactopyranoside ("IPTG") is then added to
a final concentration of 1 mM to induce transcription from the lac
repressor sensitive promoter, by inactivating the lacd repressor.
Cells subsequently are incubated further for 3 to 4 hours. Cells
then are harvested by centrifugation.
The cells are then stirred for 3-4 hours at 4.degree. C. in 6M
guanidine-HCl, pH 8. The cell debris is removed by centrifugation,
and the supernatant containing the follistatin-3 is loaded onto a
nickel-nitrilo-tri-acetic acid ("Ni-NTA") affinity resin column
(QIAGEN, Inc., supra). Proteins with a 6.times. His tag bind to the
Ni-NTA resin with high affinity and can be purified in a simple
one-step procedure (for details see: The QIAexpressionist, 1995,
QIAGEN, Inc., sipra). Briefly the supernatant is loaded onto the
column in 6 M guanidine-HCl; pH 8, the column is first washed with
10 volumes of 6 M guanidine-HCl, pH 8, then washed with 10 volumes
of 6 M guanidine-HCl pH 6, and finally the follistatin-3 is eluted
with 6 M guanidine-HCl, pH 5.
The purified protein is then renatured by dialyzing it against
phosphate-buffered saline (PBS) or 50 mM Na-acetate, pH 6 buffer
plus 200 mM NaCl. Alternatively, the protein can be successfully
refolded while immobilized on the Ni-NTA column. The recommended
conditions are as follows: renature using a linear 6M-1M urea
gradient in 500 mM NaCl, 20% glycerol, 20 mM Tris/HCl pH 7.4,
containing protease inhibitors. The renaturation should be
performed over a period of 1.5 hours or more. After renaturation
the proteins can be eluted by the addition of 250 mM immidazole.
Immidazole is removed by a final dialyzing step against PBS or 50
mM sodium acetate pH 6 buffer plus 200 mM NaCl. The purified
protein is stored at 4.degree. C. or frozen at -80.degree. The
following alternative method may be used to purify follistatin-3
expressed in E coli when it is present in the form of inclusion
bodies. Unless otherwise specified, all of the following steps are
conducted at 4-10.degree. C.
Upon completion of the production phase of the E. coli
fermentation, the cell culture is cooled to 4-10.degree. C. and the
cells are harvested by continuous centrifugation at 15,000 rpm
(Heraeus Sepatech). On the basis of the expected yield of protein
per unit weight of cell paste and the amount of purified protein
required, an appropriate amount of cell paste, by weight, is
suspended in a buffer solution containing 100 mM Tris, 50 mM EDTA,
pH 7.4. The cells are dispersed to a homogeneous suspension using a
high shear mixer.
The cells ware then lysed by passing the solution through a
microfluidizer (Microfuidics, Corp. or APV Gaulin, Inc.) twice at
4000-6000 psi. The homogenate is then mixed with NaCl solution to a
final concentration of 0.5 M NaCl, followed by centrifugation at
7000.times.g for 15 min. The resultant pellet is washed again using
0.5M NaCl, 100 mM Tris, 50 mM EDTA, pH 7.4.
The resulting washed inclusion bodies are solubilized with 1.5 M
guanidine hydrochloride (GuHCl) for 2-4 hours. After 7000.times.g
centrifugation for 15 min., the pellet is discarded and the
follistatin-3 polypeptide-containing supernatant is incubated at
4.degree. C. overnight to allow further GuHCl extraction.
Following high speed centrifugation (30,000.times.g) to remove
insoluble particles, the GuHCl solubilized protein is refolded by
quickly mixing the GuHCl extract with 20 volumes of buffer
containing 50 mM sodium, pH 4.5, 150 mM NaCl, 2 mM EDTA by vigorous
stirring. The refolded diluted protein solution is kept at
4.degree. C. without mixing for 12 hours prior to further
purification steps.
To clarify the refolded follistatin-3 polypeptide solution, a
previously prepared tangential filtration unit equipped with 0.16
.mu.m membrane filter with appropriate surface area (e.g.,
Filtron), equilibrated with 40 mM sodium acetate, pH 6.0 is
employed. The filtered sample is loaded onto a cation exchange
resin (e.g., Poros HS-50, Perseptive Biosystems). The column is
washed with 40 MM sodium acetate, pH 6.0 and eluted with 250 mM,
500 mM, 1000 mM, and 1500 mM NaCl in the same buffer, in a stepwise
manner. The absorbance at 280 mm of the effluent is continuously
monitored. Fractions are collected and further analyzed by
SDS-PAGE.
Fractions containing the follistatin-3 polypeptide are then pooled
and mixed with 4 volumes of water. The diluted sample is then
loaded onto a previously prepared set of tandem columns of strong
anion (Poros HQ-50, Perseptive Biosystems) and weak anion (Poros
CM-20, Perseptive Biosystems) exchange resins. The columns are
equilibrated with 40 mM sodium acetate, pH 6.0. Both columns are
washed with 40-mM sodium acetate, pH 6.0, 200 mM NaCl. The CM-20
column is then eluted using a 10 column volume linear gradient
ranging from 0.2 M NaCl, 50 MM sodium acetate, pH 6.0 to 1.0 M
NaCl, 50 mM sodium acetate, pH 6.5. Fractions are collected under
constant A.sub.280 monitoring of the effluent. Fractions containing
the follistatin-3 polypeptide (determined, for instance, by 16%
SDS-PAGE) are then pooled.
The resultant follistatin-3 polypeptide exhibits greater than 95%
purity after the above refolding and purification steps. No major
contaminant bands are observed from Commassie blue stained 16%
SDS-PAGE gel when 5 .mu.g of purified protein is loaded. The
purified protein is also tested for endotoxin/LPS contamination,
and typically the LPS content is less than 0.1 .mu.g/ml according
to LAL assays.
Example 2
Cloning and Expression of Follistatin-3 protein in a Baculovirus
Expression System
In this illustrative example, the plasmid shuttle vector pA2 is
used to insert the cloned DNA encoding complete protein, including
its naturally associated secretory signal (leader) sequence, into a
baculovirus to express the mature follistatin-3 protein, using
standard methods as described by Summers and colleagues (A Manual
of Methods for Baculovirus Vectors and Insect Cell Culture
Procedures, Texas Agricultural Experimental Station Bulletin No.
1555 (1987)). This expression vector contains the strong polyhedrin
promoter of the Autographa californica nuclear polyhedrosis virus
(AcMNPV) followed by convenient restriction sites such as Bam HI,
Xba I and Asp 718. The polyadenylation site of the simian virus 40
("SV40") is used for efficient polyadenylation. For easy selection
of recombinant virus, the plasmid contains the beta-galactosidase
gene from E. coli under control of a weak Drosophila promoter in
the same orientation, followed by the polyadenylation signal of the
polyhedrin gene. The inserted genes are flanked on both sides by
viral sequences for cell-mediated homologous recombination with
wild-type viral DNA to generate a viable virus that express the
cloned polynucleotide.
Many other baculovirus vectors could be used in place of the vector
above, such as pAc373, pVL941 and pAcIMI, as one skilled in the art
would readily appreciate, as long as the construct provides
appropriately located signals for transcription, translation,
secretion and the like, including a signal peptide and an in-frame
AUG as required. Such vectors are described, for instance, by
Luckow and coworkers (Virology 170:31-39 (1989)).
The cDNA sequence encoding the full length follistatin-3 protein in
the deposited clone, including the AUG initiation codon and the
naturally associated leader sequence shown in SEQ ID NO:2, is
amplified using PCR oligonucleotide primers corresponding to the 5'
and 3' sequences of the gene. The 5' primer has the sequence 5' CAT
CGC GGA TCC GCC ATC ATG CGT CCC GGG GCG CCA GGG C 3' (SEQ ID NO:14)
containing the underlined Bam HI restriction enzyme site, an
efficient signal for initiation of translation in eukaryotic cells
(Kozak, M., J. Mol. Biol. 196:947-950 (1987)), followed by 22 of
nucleotides of the sequence of the complete follistatin-3 protein
shown in FIG. 1A, beginning with the AUG initiation codon. The 3'
primer has the sequence 5' CAT CCG GGT ACC TCA CAC GAA GTT CTC TTC
CTC TTC TG 3' (SEQ ID NO:15) containing the underlined Asp 718
restriction site followed by 23 nucleotides complementary to the 3'
noncoding sequence in FIG. 1A.
The amplified fragment is isolated from a 1% agarose gel using a
commercially available kit ("Geneclean," BIO 101 Inc., La Jolla,
Calif.). The fragment then is digested with Bam HI and Asp 718 and
again is purified on a 1% agarose gel. This fragment is designated
herein F1.
The plasmid is digested with the restriction enzymes Bam HI and Asp
718 and optionally, can be dephosphorylated using calf intestinal
phosphatase, using routine procedures known in the art. The DNA is
then isolated from a 1% agarose gel using a commercially available
kit ("Geneclean" BIO 101 Inc., La Jolla, Calif.). This vector DNA
is designated herein "V1".
Fragment F1 and the dephosphorylated plasmid V1 are ligated
together with T4 DNA ligase. E. coli HB101 or other suitable E.
coli hosts such as XL-1 Blue (Statagene Cloning Systems, La Jolla,
Calif.) cells are transformed with the ligation mixture and spread
on culture plates. Bacteria are identified that contain the plasmid
with the human follistatin-3 gene by digesting DNA from individual
colonies using Bam HI and Asp 718 and then analyzing the digestion
product by gel electrophoresis. The sequence of the cloned fragment
is confirmed by DNA sequencing. This plasmid is designated herein
pA2Follistatin-3.
Five .mu.g of the piasmid pA2Follistatin-3 is co-transfected with
1.0 .mu.g of a commercially available lincartaed baculovirus DNA
("BaculoGold.TM. baculovirus DNA". Pharmiagen. San Diego. Calif.),
using the lipofeetion method described by Felgner and colleaguew
(Proc. Natl. Acad. Sci. USA 84:7413-7417 (1987)). One .mu.g of
BaculoGold.TM. virus DNA and 5 .mu.g of the plasmid
pA2Follistatin-3 are mixed in a sterile well of a microtiter plate
containing 50 .mu.l of serum-free Graces medium (Life Technologies
Inc., Gaitbersburg, Md.). Afterwards, 10 .mu.l Lipofecun plus 90
.mu.l Grace's medium are added, mixed and incubated for 15 minutes
room temperature. Then the transfeccion mixture is added drop-wise
to Sf9 insect cells (ATCC.RTM. CRL 1711) seeded in a 35 mm tissue
culture plate with 1 ml Grace's medium without serum. The plate is
then incubated for 5 hours at 27.degree. C. The transfection
solution is then removed from the plate and 1 ml of Grace's insect
medium supplemented with 10% fetal calf serum is added. Cultivation
is then continued at 27.degree. C. for four days.
After four days the supernatant is collected and a plaque assay is
performed, as described by Summers and Smith (supra). An agarose
gel with "Blue Gal" (Life Technologies Inc., Gaithersburg) is used
to allow easy identification and isolation of gal-expressing
clones, which produce blue-stained plaques. (A detailed description
of a "plaque assay" of this type can also be found in the user's
guide for insect cell culture and baculovirology distributed by
Life Technologies Inc., Gaithersburg, page 9-10). After appropriate
incubation, blue stained plaques are picked with the tip of a
micropipettor (e.g., Eppendorf). The agar containing the
recombinant viruses is then resuspended in a microcentrifuge tube
containing 200 .mu.l of Grace's medium and the suspension
containing the recombinant baculovirus is used to infect Sf9 cells
seeded in 35 mm dishes. Four days later the supernatants of these
culture dishes are harvested and then they are stored at 4.degree.
C. The recombinant virus is called V-Follistatin-3.
To verify the expression of the follistatin-3 gene Sf9 cells are
grown in Grace's medium supplemented with 10% heat-inactivated FBS.
The cells are infected with the recombinant baculovirus
V-Follistatin-3 at a multiplicity of infection ("MOI") of about 2.
If radiolabeled proteins are desired, 6 hours later the medium is
removed and is replaced with SF900 II medium minus methionine and
cysteine (available from Life Technologies Inc., Rockville, Md.).
After 42 hours, 5 .mu.Ci of .sup.35 S-methionine and 5 .mu.Ci
.sup.35 S-cysteine (available from Amersham) are added. The cells
are further incubated for 16 hours and then are harvested by
centrifugation. The proteins in the supernatant as well as the
intracellular proteins are analyzed by SDS-PAGE followed by
autoradiography (if radiolabeled).
Microsequencing of the amino acid sequence of the amino terminus of
purified protein may be used to determine the amino terminal
sequence of the mature form of the follistatin-3 protein, and thus
the cleavage point and length of the naturally associated secretory
signal peptide.
Follistatin-3 protein has been produced by the above described
process in a baculovirus expression system. The resultant
follistatin-3 polypeptide was isolated and C-terminal sequencing
analysis was used to confirm the prediction that the N-terminal 26
amino acids of the full-length follistatin-3 polypeptide shown in
FIGS. 1A, 1B, and 1C (and in SEQ ID NO:2) are cleaved and that the
mature form of the follistatin-3 polypeptide begins with
methionine-27 as the N-terminal residue according to the numbering
scheme of FIGS. 1A, 1B, and 1C (which is identical to methionine-1
according to the numbering scheme of SEQ ID NO:2). Of course, it is
important to remember that the observed mature form of a secreted
protein may vary according to a number of factors as detailed
above.
Example 3
Cloning and Expression of Follistatin-3 in Mammalian Cells
A typical mammalian expression vector contains the promoter
clement, which mediates the initiation of transcription of mRNA,
the protein coding sequence, and signals required for the
termination of transcription and polyadenylation of the transcript.
Additional elements include enhancers, Kozak sequences and
intervening sequences flanked by donor and acceptor sites for RNA
splicing. Highly efficient transcription can be achieved with the
early and late promoters from SV40, the long terminal repeats
(LTRs) from Retroviruses, e.g., RSV, HTVLI, HIVI and the early
promoter of the cycomegalovirus (CMV). However, cellular elements
can also be used (e.g., the human actin promoter). Suitable
expression vectors for use in practicing the present invention
include, for example, vectors such as pSVL and pMSG (Pharmacia.
Uppsala, Sweden), pRSVcar (ATCC.RTM. 37152), pSV2dhfr (ATCC.RTM.
37146) and pBC12MI (ATCC.RTM. 67102). Mammalian host cells that
could be used include, human Hela, 293. H9 and Jurkat cells, moUse
NIH3T3 and C127 cells, Cos 1, Cos 7 and CV1, quail QC1-3 cells,
mouse L cells and Chinese hamster ovary (CHO) cells.
Alternatively, the gene can be expressed in stable cell lines that
contain the gene integrated into a chromosome. The co-transfection
with a selectable marker such as dhfr, gpt, neomycin, hygromycin
allows the identification and isolation of the transfected
cells.
The transfected gene can also be amplified to express large amounts
of the encoded protein. The DHFR (dihydrofolate reductase) marker
is useful to develop cell lines that carry several hundred or even
several thousand copies of the gene of interest. Another useful
selection marker is the enzyme glutamine synthase (GS; Murphy, et
al., Biochem J. 227:277-279 (1991); Bebbington, et al.,
Bio/Technology 10:169-175 (1992)). Using these markers, the
mammalian cells are grown in selective medium and the cells with
the highest resistance are selected. These cell lines contain the
amplified gene(s) integrated into a chromosome. Chinese hamster
ovary (CHO) and NSO cells are often used for the production of
proteins.
The expression-vectors pC1 and pC4 contain the strong promoter
(LTR) of the Rous Sarcoma Virus (Cullen, et al., Mol. Cel. Biol.
5:438-447 (1985)) plus a fragment of the CMV-enhancer (Boshart, et
al., Cell 41:521-530 (1985)). Multiple cloning sites, e.g., with
the restriction enzyme cleavage sites Bam HI, Xba I and Asp 718,
facilitate the cloning of the gene of interest. The vectors contain
in addition the 3' intron, the polyadenylation and termination
signal of the rat preproinsulin gene.
Example 3(a)
Cloning and Expression in COS Cells
The expression plasmid, pFollistatin-3HA, is made by cloning a
portion of the cDNA encoding the mature form of the follistatin-3
protein into the expression vector pcDNAI/Amp or pcDNAIII (which
can be obtained from Invitrogen, Inc.).
The expression vector pcDNAI/amp contains: (1) an E. coli origin of
replication effective for propagation in E. coli and other
prokaryotic cells; (2) an ampicillin resistance gene for selection
of plasmid-containing prokaryotic cells; (3) an SV40 origin of
replication for propagation in eukaryotic cells; (4) a CIV
promoter, a polylinker, an SV40 intron; (5) several codons encoding
a hemagglutinin fragment (i.e., an "HA" tag to facilitate
purification) followed by a termination codon and polyadenylation
signal arranged so that a cDNA can be conveniently placed under
expression control of the CMV promoter and operably linked to the
SV40 intron and the polyadenylation signal by means of restriction
sites in the polylinker. The HA tag corresponds to an epitope
derived from the influenza hemagglutinin protein described by
Wilson and colleagues (Cell 37:767 (1984)). The fusion of the HA
tag to the target protein allows easy detection and recovery of the
recombinant protein with an antibody that recognizes the HA
epitope. pcDNAIII contains, in addition, the selectable neomycin
marker. A DNA fragment encoding the complete follistatin-3
polypeptide is cloned into the polylinker region of the vector so
that recombinant protein expression is directed by the CMV
promoter. The plasmid construction strategy is as follows. The
follistatin-3 cDNA of the deposited clone is amplified using
primers that contain convenient restriction sites, much as
described above for construction of vectors for expression of
follistatin-3 in E. coli. Suitable primers include the following,
which are used in this example. The 5' primer, containing the
underlined Baam HI site, a Kozak sequence, an AUG start codon, and
22 nucleotides of the 5' coding region of the complete
follistatin-3 polypeptide, has the following sequence: 5' CAT CGC
GGA TCC GCC ACC ATG CGT CCC GGG GCG CCA GGG C 3' (SEQ ID NO:16).
The 3' primer, containing the underlined Asp 718 and 23 of
nucleotides complementary to the 3' coding sequence immediately
before the-stop codon, has the following sequence: 5' TCA CCG CTC
GAG CAC GAA GTT CTC TTC CTC TTC TG 3' (SEQ ID NO:17).
The PCR amplified DNA fragment and the vector, pcDNAI/Amp, are
digested with Bam HI and Asp 718 and then ligated. The ligation
mixture is transformed into E. coli strain SURE (Stratagene Cloning
Systems, La Jolla, Calif. 92037), and the transformed culture is
plated on ampicillin media plates which then are incubated to allow
growth of ampicillin resistant colonies. Plasmid DNA is isolated
from resistant colonies and examined by restriction analysis or
other means for the presence of the fragment encoding the complete
follistatin-3 polypeptide For expression of recombinant
follistatin-3, COS cells are transfected with an expression vector,
as described above, using DEAE-dextran, as described, for instance,
by Sambrook and coworkers (Molecular Cloning: a Laboratory Manual,
Cold Spring Laboratory Press, Cold Spring Harbor, N.Y. (1989)).
Cells are incubated under conditions for expression of
follistatin-3 by the vector.
Expression of the follistatin-3-HA fusion protein is detected by
radiolabeling and immunoprecipitation, using methods described in,
for example Harlow and colleagues (Antibodies: A Laboratory Manual,
2nd Ed.; Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
N.Y. (1988)). To this end, two days after transfection, the cells
are labeled by incubation in media containing .sup.35 S-cysteine
for 8 hours. The cells and the media are collected, and the cells
are washed and the lysed with detergent-containing RIPA buffer: 150
mM NaCl, 1% NP-40, 0.1% SDS, 1% NP-40, 0.5% DOC, 50 mM TRIS, pH
7.5, as described by Wilson and colleagues (supra). Proteins are
precipitated from the cell lysate and from the culture media using
an HA-specific monoclonal antibody. The precipitated proteins then
are analyzed by SDS-PAGE and autoradiography. An expression product
of the expected size is seen in the cell lysate, which is not seen
in negative controls.
Example 3(b)
Cloning and Expression in CHO Cells
The vector pC4 is used for the expression of follistatin-3
polypeptde. Plasmid pC4 is a derivative of the plasmid pSV2-dhfr
(ATCC.RTM. Accession No. 37146) The plasmid contains the mouse DHFR
gene under control of the SV40 early promoter. Chinese hamster
ovary- or other cells lacking dihydrofolart activity that are
transfected with these placmids can be selected by growing the
cells in a selective medium (alpha minus MEM,. Life Technologies)
supplemented With the chemotherapuric agent methotrexate. The
amplification of the DHFR genes in cells resistant to methotrexate
(MTX) has ben well documented (see, e.g., Alt, F. W., et al., J.
Biol. Chem. 253:1357-1370(1978); Hamlin, J. L. and Ma, C. Biochem.
et Biophys. Acta, 1097:107-143 (1990); Page, M. J. and Sydenham, M.
A. Biotechnology 9:64-68 (1991)). Cells grown in iocreasing
concentrations of MTX develop resistance to the drug by
overproducing the target enzyme, DHFR, as a result of amplification
of the DHFR gene. If a second gene is linked to the DHFR gene, it
is usually co-amplified and over-expressed. It is known in the art
that this approach may be used to develop cell lines carrying more
than 1,000 copies of the amplified gene(s). Subsequently, when the
methotrexate is withdrawn, cell lines are obtained which contain
the amplified gene integrated into one or more chromosome(s) of the
host cell.
Plasmid pC4 contains for expressing the gene of interest the strong
promoter of the long terminal repeat (LTR) of the Rouse Sarcoma
Virus (Cullen, et al., Mol. Cell. Biol. 5:438-447 (1985)) plus a
fragment isolated from the enhancer of the immediate early gene of
human cytomegalovirus (CMV; Boshart, et al., Cell 41:521-530
(1985)). Downstream of the promoter are the following single
restriction enzyme cleavage sites that allow the integration of the
genes: Bam HI, Xba I, and Asp 718. Behind these cloning sites the
plasmid contains the 3' intron and polyadenylation site of the rat
preproinsulin gene. Other high efficiency promoters can also be
used for the expression, e.g., the human B-actin promoter, the SV40
early or late promoters or the long terminal repeats from other
retroviruses, e.g., HIV and HTLVI. Clontech's Tet-Off and Tet-On
gene expression systems and similar systems can be used to express
the follistatin-3 polypeptide in a regulated way in mammalian cells
(Gossen, M., and Bujard, H. Proc. Natl. Acad. Sci. USA 89:5547-5551
(1992)). For the polyadenylation of the mRNA other signals, e.g.,
from the human growth hormone or globin genes can be used as well.
Stable cell lines carrying a gene of interest integrated into the
chromosomes can also be selected upon co-transfection with a
selectable marker such as gpt, G418 or hygromycin. It is
advantageous to use more than one selectable marker in the
beginning, e.g., G418 plus methotrexate.
The plasmid pC4 is digested with the restriction enzymes Bam HI and
Asp 718 and then dephosphorylated using calf intestinal phosphates
by procedures known in the art. The vector is then isolated from a
1% agarose gel. The DNA sequence encoding the complete
follistatin-3 polypeptide is amplified using PCR oligonucleotide
primers corresponding to the 5' and 3' sequences of the desired
portion of the gene. The 5' primer containing the underlined Bam HI
site, a Kozak sequence, an AUG start codon, and 22 nucleotides of
the 5' coding region of the complete follistatin-3 polypeptide, has
the following sequence: 5' CAT CGC GGA TCC GCC ACC ATG CGT CCC GGG
GCG CCA GGG C 3' (SEQ ID NO:18). The 3' primer, containing the
underlined Asp 718 restriction site and 23 of nucleotides
complementary to the 3' coding sequence immediately before the stop
codon as shown in FIG. 1A (SEQ ID NO:1), has the following
sequence: 5' CAT CCG GGT ACC TCA CAC GAA GTT CTC TTC CTC TTC TG 3'
(SEQ ID NO:19).
The amplified fragment is digested with the endonucleases Bam HI
and Asp 718 and then purified again on a 1% agarose gel. The
isolated fragment and the dephosphorylated vector are then ligated
with T4 DNA ligase. E. coli HB 101 or XL-1 Blue cells are then
transformed and bacteria are identified that contain the fragment
inserted into plasmid pC4 using, for instance, restriction enzyme
analysis.
Chinese hamster ovary cells lacking an active DHFR gene are used
for transfection. Five .mu.g of the expression plasmid pC4 is
cotransfected with 0.5 .mu.g of the plasmid pSVneo using lipofectin
(Felgner, et al., sitpra). The plasmid pSV2-neo contains a dominant
selectable marker, the neo gene from Tn5 encoding an enzyme that
confers resistance to a group of antibiotics including G418. The
cells are seeded in alpha minus MEM supplemented with 1 mg/ml G418.
After 2 days, the cells are trypsinized and seeded in hybridoma
cloning plates (Greiner, Germany) in alpha minus MEM supplemented
with 10, 25, or 50 ng/ml of metothrexate plus 1 mg/ml G418. After
about 10-14 days single clones are trypsinized and then seeded in
6-well petri dishes or 10 ml flasks using different concentrations
of methotrexate (50 nM, 100 nM, 200 nM, 400 nM, 800 nM). Clones
growing at the highest concentrations of methotrexate are then
transferred to new 6-well plates containing even higher
concentrations of methotrexate (1 .mu.M, 2 .mu.M, 5 .mu.M, 10 mM,
20 mM). The same procedure is repeated until clones are obtained
which grow at a concentration of 100-200 .mu.M. Expression of the
desired gene product is analyzed, for instance, by SDS-PAGE and
Western blot or by reversed phase HPLC analysis.
Follistatin-3 protein has been produced by the abovedescribed
process in a CHO cell expression system. The resultant
follistatin-3 polypeptide was isolated and C-terminal sequencing
analysis was used to confirm the prediction that the N-terminal 26
amino acids of the full-length follistatin-3 polypeptide shown in
FIGS. 1 A, 1B, and 1C (and in SEQ ID NO:2) are cleaved and that the
mature form of the follistatin-3 polypeptide begins with
methionine-27 as the N-terminal residue according to the numbering
scheme of FIGS. 1A, 1B, and 1C (which is identical to methionine-1
according to the numbering scheme of SEQ ID NO:2). Of course, it is
important to remember that the observed mature form of a secreted
protein may vary according to a number of factors as detailed
above.
Example 4
Tissue Distribution of Follistatin-3 mRNA Expression
Northern blot analysis was carried out to examine follistatin-3
gene expression in human tissues, using methods described by, among
others, Sambrook and colleagues (supra). A cDNA probe containing
the entire nucleotide sequence of the follistatin-3 protein (SEQ ID
NO:1) was labeled with .sup.32 p using the rediprime.TM. DNA
labeling system (Amersham Life Science), according to
manufacturer's instructions. After labeling, the probe was purified
using a CHROMA SPIN-100.TM. column (Clontech Laboratories, Inc.),
according to manufacturer's protocol number PT1200-1. The purified
labeled probe was then used to examine various human tissues for
follistatin-3 mRNA.
Multiple Tissue Northern (MTN) blots containing various human
tissues (H) or human immune system tissues (IM) were obtained from
Clontech and were examined with the labeled probe using
ExpressHyb.TM. hybridization solution (Clontech) according to
manufacturer's protocol number PT1190-1. Following hybridization
and washing, the blots were mounted and exposed to film at
-70.degree. C. overnight, and films developed according to standard
procedures. The follistatin-3-specific probe recognized an mRNA
species of approximately 2.6 kb in most tissues examined.
It will be clear that the invention may be practiced otherwise than
as particularly described in the foregoing description and
examples. Numerous modifications and variations of the present
invention are possible in light of the above teachings and,
therefore, are within the scope of the appended claims.
The entire disclosure of all publications (including patents,
patent applications, journal articles, laboratory manuals, books,
or other documents) cited herein are hereby incorporated by
reference.
Further, the Sequence Listing submitted herewith, and the Sequence
Listing submitted with U.S. Provisional Application Serial No.
60/056,248, filed on Aug. 29, 1997 (to which the present
application claims benefit of the filing date under 35 U.S.C.
.sctn. 119(e)), in both computer and paper forms are hereby
incorporated by reference in their entireties.
SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 19 <210>
SEQ ID NO 1 <211> LENGTH: 2495 <212> TYPE: DNA
<213> ORGANISM: Homo sapiens <220> FEATURE: <221>
NAME/KEY: CDS <222> LOCATION: (19)..(807) <221>
NAME/KEY: mat_peptide <222> LOCATION: (97)..(807) <221>
NAME/KEY: sig_peptide <222> LOCATION: (19)..(96) <221>
NAME/KEY: misc_feature <222> LOCATION: (2429) <223>
OTHER INFORMATION: n equals a, t, g, or c <400> SEQUENCE: 1
gccgtctctg cgttcgcc atg cgt ccc ggg gcg cca ggg cca ctc tgg cct 51
Met Arg Pro Gly Ala Pro Gly Pro Leu Trp Pro -25 -20 ctg ccc tgg ggg
gcc ctg gct tgg gcc gtg ggc ttc gtg agc tcc atg 99 Leu Pro Trp Gly
Ala Leu Ala Trp Ala Val Gly Phe Val Ser Ser Met -15 -10 -5 -1 1 ggc
tcg ggg aac ccc gcg ccc ggt ggt gtt tgc tgg ctc cag cag ggc 147 Gly
Ser Gly Asn Pro Ala Pro Gly Gly Val Cys Trp Leu Gln Gln Gly 5 10 15
cag gag gcc acc tgc agc ctg gtg ctc cag act gat gtc acc cgg gcc 195
Gln Glu Ala Thr Cys Ser Leu Val Leu Gln Thr Asp Val Thr Arg Ala 20
25 30 gag tgc tgt gcc tcc ggc aac att gac acc gcc tgg tcc aac ctc
acc 243 Glu Cys Cys Ala Ser Gly Asn Ile Asp Thr Ala Trp Ser Asn Leu
Thr 35 40 45 cac ccg ggg aac aag atc aac ctc ctc ggc ttc ttg ggc
ctt gtc cac 291 His Pro Gly Asn Lys Ile Asn Leu Leu Gly Phe Leu Gly
Leu Val His 50 55 60 65 tgc ctt ccc tgc aaa gat tcg tgc gac ggc gtg
gag tgc ggc ccg ggc 339 Cys Leu Pro Cys Lys Asp Ser Cys Asp Gly Val
Glu Cys Gly Pro Gly 70 75 80 aag gcg tgc cgc atg ctg ggg ggc cgc
ccg cgc tgc gag tgc gcg ccc 387 Lys Ala Cys Arg Met Leu Gly Gly Arg
Pro Arg Cys Glu Cys Ala Pro 85 90 95 gac tgc tcg ggg ctc ccg gcg
cgg ttg cag gtc tgc ggc tca gac ggc 435 Asp Cys Ser Gly Leu Pro Ala
Arg Leu Gln Val Cys Gly Ser Asp Gly 100 105 110 gcc acc tac cgc gac
gag tgc gag ctg cgc gcc gcg cgc tgc cgc ggc 483 Ala Thr Tyr Arg Asp
Glu Cys Glu Leu Arg Ala Ala Arg Cys Arg Gly 115 120 125 cac ccg gac
ctg agc gtc atg tac cgg ggc cgc tgc cgc aag tcc tgt 531 His Pro Asp
Leu Ser Val Met Tyr Arg Gly Arg Cys Arg Lys Ser Cys 130 135 140 145
gag cac gtg gtg tgc ccg cgg cca cag tcg tgc gtc gtg gac cag acg 579
Glu His Val Val Cys Pro Arg Pro Gln Ser Cys Val Val Asp Gln Thr 150
155 160 ggc agc gcc cac tgc gtg gtg tgt cga gcg gcg ccc tgc cct gtg
ccc 627 Gly Ser Ala His Cys Val Val Cys Arg Ala Ala Pro Cys Pro Val
Pro 165 170 175 tcc agc ccc ggc cag gag ctt tgc ggc aac aac aac gtc
acc tac atc 675 Ser Ser Pro Gly Gln Glu Leu Cys Gly Asn Asn Asn Val
Thr Tyr Ile 180 185 190 tcc tcg tgc cac atg cgc cag gcc acc tgc ttc
ctg ggc cgc tcc atc 723 Ser Ser Cys His Met Arg Gln Ala Thr Cys Phe
Leu Gly Arg Ser Ile 195 200 205 ggc gtg cgc cac gcg ggc agc tgc gca
ggc acc cct gag gag ccg cca 771 Gly Val Arg His Ala Gly Ser Cys Ala
Gly Thr Pro Glu Glu Pro Pro 210 215 220 225 ggt ggt gag tct gca gaa
gag gaa gag aac ttc gtg tgagcctgca 817 Gly Gly Glu Ser Ala Glu Glu
Glu Glu Asn Phe Val 230 235 ggacaggcct gggcctggtg cccgaggccc
cccatcatcc cctgttattt attgccacag 877 cagagtctaa tttatatgcc
acggacactc cttagagccc ggattcggac cacttgggga 937 tcccagaacc
tccctgacga tatcctggaa ggactgagga agggaggcct gggggccggc 997
tggtgggtgg gatagacctg cgttccggac actgagcgcc tgatttaggg cccttctcta
1057 ggatgcccca gcccctaccc taagacctat tgccggggag gattccacac
ttccgctcct 1117 ttggggataa acctattaat tattgctact atcaagaggg
ctgggcattc tctgctggta 1177 attcctgaag aggcatgact gcttttctca
gccccaagcc tctagtctgg gtgtgtacgg 1237 agggtctagc ctgggtgtgt
acggagggtc tagcctgggt gagtacggag ggtctagcct 1297 gggtgagtac
ggaggatcta gcctgggtga gtacggagag tctagcctgg gtgtgtatgg 1357
aggatctagc ctgggtgagt atggagggtc tagcctgggt gagtatggag ggtctagcct
1417 gggtgtgtat ggagggtcta gcctgggtga gtatggaggg tctagcctgg
gtgtgtatgg 1477 agggtctagc ctgggtgagt atggagggtc tagcctgggt
gtgtacggag ggtctagtct 1537 gagtgcgtgt ggggacctca gaacactgtg
accttagccc agcaagccag gcccttcatg 1597 aaggccaaga aggctgccac
cattccctgc cagcccaaga actccagctt ccccactgcc 1657 tctgtgtgcc
cctttgcgtc ctgtgaaggc cattgagaaa tgcccagtgt gccccctggg 1717
aaagggcacg gcctgtgctc ctgacacggg ctgtgcttgg ccacagaacc acccagcgtc
1777 tcccctgctg ctgtccacgt cagttcatga ggcaacgtcg cgtggtctca
gacgtggagc 1837 agccagcggc agctcagagc agggcactgt gtccggcgga
gccaagtcca ctctggggga 1897 gctctggcgg ggaccacggg ccactgctca
cccactggcc ccgagggggg tgtagacgcc 1957 aagactcacg catgtgtgac
atccggagtc ctggagccgg gtgtcccagt ggcaccacta 2017 ggtgcctgct
gcctccacag tggggttcac acccagggct ccttggtccc ccacaacctg 2077
ccccggccag gcctgcagac ccagactcca gccagacctg cctcacccac caatgcagcc
2137 ggggctggcg acaccagcca ggtgctggtc ttgggccagt tctcccacga
cggctcaccc 2197 tcccctccat ctgcgttgat gctcagaatc gcctacctgt
gcctgcgtgt aaaccacagc 2257 ctcagaccag ctatggggag aggacaacac
ggaggatatc cagcttcccc ggtctggggt 2317 gaggagtgtg gggagcttgg
gcatcctcct ccagcctcct ccagccccca ggcagtgcct 2377 tacctgtggt
gcccagaaaa gtgcccctag gttggtgggt ctacaggagc cncagccagg 2437
cagcccaccc caccctgggg ccctgcctca ccaaggaaat aaagactcaa agaagcct
2495 <210> SEQ ID NO 2 <211> LENGTH: 263 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
2 Met Arg Pro Gly Ala Pro Gly Pro Leu Trp Pro Leu Pro Trp Gly Ala
-25 -20 -15 Leu Ala Trp Ala Val Gly Phe Val Ser Ser Met Gly Ser Gly
Asn Pro -10 -5 -1 1 5 Ala Pro Gly Gly Val Cys Trp Leu Gln Gln Gly
Gln Glu Ala Thr Cys 10 15 20 Ser Leu Val Leu Gln Thr Asp Val Thr
Arg Ala Glu Cys Cys Ala Ser 25 30 35 Gly Asn Ile Asp Thr Ala Trp
Ser Asn Leu Thr His Pro Gly Asn Lys 40 45 50 Ile Asn Leu Leu Gly
Phe Leu Gly Leu Val His Cys Leu Pro Cys Lys 55 60 65 70 Asp Ser Cys
Asp Gly Val Glu Cys Gly Pro Gly Lys Ala Cys Arg Met 75 80 85 Leu
Gly Gly Arg Pro Arg Cys Glu Cys Ala Pro Asp Cys Ser Gly Leu 90 95
100 Pro Ala Arg Leu Gln Val Cys Gly Ser Asp Gly Ala Thr Tyr Arg Asp
105 110 115 Glu Cys Glu Leu Arg Ala Ala Arg Cys Arg Gly His Pro Asp
Leu Ser 120 125 130 Val Met Tyr Arg Gly Arg Cys Arg Lys Ser Cys Glu
His Val Val Cys 135 140 145 150 Pro Arg Pro Gln Ser Cys Val Val Asp
Gln Thr Gly Ser Ala His Cys 155 160 165 Val Val Cys Arg Ala Ala Pro
Cys Pro Val Pro Ser Ser Pro Gly Gln 170 175 180 Glu Leu Cys Gly Asn
Asn Asn Val Thr Tyr Ile Ser Ser Cys His Met 185 190 195 Arg Gln Ala
Thr Cys Phe Leu Gly Arg Ser Ile Gly Val Arg His Ala 200 205 210 Gly
Ser Cys Ala Gly Thr Pro Glu Glu Pro Pro Gly Gly Glu Ser Ala 215 220
225 230 Glu Glu Glu Glu Asn Phe Val 235 <210> SEQ ID NO 3
<211> LENGTH: 317 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 3 Met Val Arg Ala Arg His Gln
Pro Gly Gly Leu Cys Leu Leu Leu Leu 1 5 10 15 Leu Leu Cys Gln Phe
Met Glu Asp Arg Ser Ala Gln Ala Gly Asn Cys 20 25 30 Trp Leu Arg
Gln Ala Lys Asn Gly Arg Cys Gln Val Leu Tyr Lys Thr 35 40 45 Glu
Leu Ser Lys Glu Glu Cys Cys Ser Thr Gly Arg Leu Ser Thr Ser 50 55
60 Trp Thr Glu Glu Asp Val Asn Asp Asn Thr Leu Phe Lys Trp Met Ile
65 70 75 80 Phe Asn Gly Gly Ala Pro Asn Cys Ile Pro Cys Lys Glu Thr
Cys Glu 85 90 95 Asn Val Asp Cys Gly Pro Gly Lys Lys Cys Arg Met
Asn Lys Lys Asn 100 105 110 Lys Pro Arg Cys Val Cys Ala Pro Asp Cys
Ser Asn Ile Thr Trp Lys 115 120 125 Gly Pro Val Cys Gly Leu Asp Gly
Lys Thr Tyr Arg Asn Glu Cys Ala 130 135 140 Leu Leu Lys Ala Arg Cys
Lys Glu Gln Pro Glu Leu Glu Val Gln Tyr 145 150 155 160 Gln Gly Arg
Cys Lys Lys Thr Cys Arg Asp Val Phe Cys Pro Gly Ser 165 170 175 Ser
Thr Cys Val Val Asp Gln Thr Asn Asn Ala Tyr Cys Val Thr Cys 180 185
190 Asn Arg Ile Cys Pro Glu Pro Ala Ser Ser Glu Gln Tyr Leu Cys Gly
195 200 205 Asn Asp Gly Val Thr Tyr Ser Ser Ala Cys His Leu Arg Lys
Ala Thr 210 215 220 Cys Leu Leu Gly Arg Ser Ile Gly Leu Ala Tyr Glu
Gly Lys Cys Ile 225 230 235 240 Lys Ala Lys Ser Cys Glu Asp Ile Gln
Cys Thr Gly Gly Lys Lys Cys 245 250 255 Leu Trp Asp Phe Lys Val Gly
Arg Gly Arg Cys Ser Leu Cys Asp Glu 260 265 270 Leu Cys Pro Asp Ser
Lys Ser Asp Glu Pro Val Cys Ala Ser Asp Asn 275 280 285 Ala Thr Tyr
Ala Ser Glu Cys Ala Met Lys Glu Ala Ala Cys Ser Ser 290 295 300 Gly
Val Leu Leu Glu Val Lys His Ser Gly Ser Cys Asn 305 310 315
<210> SEQ ID NO 4 <211> LENGTH: 508 <212> TYPE:
DNA <213> ORGANISM: Homo sapiens <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (377)
<223> OTHER INFORMATION: n equals a, t, g, or c <221>
NAME/KEY: misc_feature <222> LOCATION: (379) <223>
OTHER INFORMATION: n equals a, t, g, or c <221> NAME/KEY:
misc_feature <222> LOCATION: (428) <223> OTHER
INFORMATION: n equals a, t, g, or c <221> NAME/KEY:
misc_feature <222> LOCATION: (439) <223> OTHER
INFORMATION: n equals a, t, g, or c <221> NAME/KEY:
misc_feature <222> LOCATION: (444) <223> OTHER
INFORMATION: n equals a, t, g, or c <221> NAME/KEY:
misc_feature <222> LOCATION: (452) <223> OTHER
INFORMATION: n equals a, t, g, or c <221> NAME/KEY:
misc_feature <222> LOCATION: (461) <223> OTHER
INFORMATION: n equals a, t, g, or c <221> NAME/KEY:
misc_feature <222> LOCATION: (466) <223> OTHER
INFORMATION: n equals a, t, g, or c <221> NAME/KEY:
misc_feature <222> LOCATION: (468) <223> OTHER
INFORMATION: n equals a, t, g, or c <221> NAME/KEY:
misc_feature <222> LOCATION: (470) <223> OTHER
INFORMATION: n equals a, t, g, or c <221> NAME/KEY:
misc_feature <222> LOCATION: (475) <223> OTHER
INFORMATION: n equals a, t, g, or c <221> NAME/KEY:
misc_feature <222> LOCATION: (485) <223> OTHER
INFORMATION: n equals a, t, g, or c <221> NAME/KEY:
misc_feature <222> LOCATION: (501) <223> OTHER
INFORMATION: n equals a, t, g, or c <221> NAME/KEY:
misc_feature <222> LOCATION: (505) <223> OTHER
INFORMATION: n equals a, t, g, or c <221> NAME/KEY:
misc_feature <222> LOCATION: (399) <223> OTHER
INFORMATION: n equals a, t, g or c <400> SEQUENCE: 4
aattcggcac gagtttctca gccccaagcc tctagtctgg gtgtgtacgg agggtctagc
60 ctgggtgtgt acggagggtc tagcctgggt gagtacggag ggtctagcct
gggtgagtac 120 ggagggtcta gcctgggtga gtacggagag tctagcctgg
gtgtgtatgg aggatctagc 180 ctgggtgagt atggagggtc tagcctgggt
gagtatggag ggtctagcct gggtgtgtat 240 ggagggtcta gcctgggtga
gtatggaggg tctagcctgg gtgtgtatgg agggtctagc 300 ctgggtgagt
atggagggtc tagccttggt gtttacggag ggtctagtct gagttcgttt 360
tggggacctc agaacantnt taacctttag cccagnaanc caggccctta atgaaggcca
420 gaaggttnca ccattcctnc cctnccaaga antcaatttc nnaatncntn
ttgtnccctt 480 ttggnccttt aagccattta naatncca 508 <210> SEQ
ID NO 5 <211> LENGTH: 466 <212> TYPE: DNA <213>
ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (415) <223> OTHER
INFORMATION: n equals a, t, g, or c <221> NAME/KEY:
misc_feature <222> LOCATION: (422)
<223> OTHER INFORMATION: n equals a, t, g, or c <221>
NAME/KEY: misc_feature <222> LOCATION: (437) <223>
OTHER INFORMATION: n equals a, t, g, or c <221> NAME/KEY:
misc_feature <222> LOCATION: (449) <223> OTHER
INFORMATION: n equals a, t, g, or c <400> SEQUENCE: 5
ggcgacggcg tggagtgcgg cccgggcaag gcgtgccgca tgctgggggg ccgcccgcgc
60 tgcgagtgcg cgcccgactg ctcggggctc ccggcgcggt tgcaggtctg
cggctcagac 120 ggcgccacct accgcgacga gtgcgagctg cgcgccgcgc
gctgccgcgg ccacccggac 180 ctgagcgtca tgtaccgggg ccgctgccgc
aagtcctgtg agcacgtggt gtgcccgcgg 240 ccacagtcgt gcgtcgtgga
ccagacgggc agcgcccact gcgtggtgtg tcgaagcggc 300 gccctgccct
gtgccctcca gccccggcca ggagctttgc ggccaacaac aaagttacct 360
aaatttcttc gtgccaaatg cgccaaggcc aactgcttcc tgggccggtt ccatnnggcg
420 tncgccaagc gggcaanttt cgcaagcanc cctgaaggag ccgcca 466
<210> SEQ ID NO 6 <211> LENGTH: 337 <212> TYPE:
DNA <213> ORGANISM: Homo sapiens <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (33)
<223> OTHER INFORMATION: n equals a, t, g, or c <221>
NAME/KEY: misc_feature <222> LOCATION: (60) <223> OTHER
INFORMATION: n equals a, t, g, or c <221> NAME/KEY:
misc_feature <222> LOCATION: (92)..(93) <223> OTHER
INFORMATION: n equals a, t, g, or c <221> NAME/KEY:
misc_feature <222> LOCATION: (141) <223> OTHER
INFORMATION: n equals a, t, g, or c <221> NAME/KEY:
misc_feature <222> LOCATION: (264) <223> OTHER
INFORMATION: n equals a, t, g, or c <221> NAME/KEY:
misc_feature <222> LOCATION: (129) <223> OTHER
INFORMATION: n equals a, t, g or c <400> SEQUENCE: 6
cttgagtgcg tgtggggacc tcagaacact gtnaccttag cccagcaagc caggcccttn
60 atgaaggcca agaaggctgc caccattccc tnncagccca agaactccag
cttccccact 120 gcctctttnt gcccctttgc ntcctgtgaa ggccattgag
aaatgcccag tgtgccccct 180 gggaaagggc acggcctgtg ctcctgacac
gggctgtgct tggccacaga accacccagc 240 gtctcccctg ctgctgtcca
cgtnagttca tgaggcaacg tcgcgtggtc ttcagacgtg 300 ggagcagcca
gcggcagctc aggaggcagg gcactgt 337 <210> SEQ ID NO 7
<211> LENGTH: 298 <212> TYPE: DNA <213> ORGANISM:
Homo sapiens <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (5) <223> OTHER
INFORMATION: n equals a, t, g, or c <221> NAME/KEY:
misc_feature <222> LOCATION: (11) <223> OTHER
INFORMATION: n equals a, t, g, or c <221> NAME/KEY:
misc_feature <222> LOCATION: (68) <223> OTHER
INFORMATION: n equals a, t, g, or c <221> NAME/KEY:
misc_feature <222> LOCATION: (70) <223> OTHER
INFORMATION: n equals a, t, g, or c <221> NAME/KEY:
misc_feature <222> LOCATION: (84) <223> OTHER
INFORMATION: n equals a, t, g, or c <221> NAME/KEY:
misc_feature <222> LOCATION: (111) <223> OTHER
INFORMATION: n equals a, t, g, or c <221> NAME/KEY:
misc_feature <222> LOCATION: (186) <223> OTHER
INFORMATION: n equals a, t, g, or c <221> NAME/KEY:
misc_feature <222> LOCATION: (243) <223> OTHER
INFORMATION: n equals a, t, g, or c <221> NAME/KEY:
misc_feature <222> LOCATION: (266) <223> OTHER
INFORMATION: n equals a, t, g, or c <221> NAME/KEY:
misc_feature <222> LOCATION: (272) <223> OTHER
INFORMATION: n equals a, t, g, or c <221> NAME/KEY:
misc_feature <222> LOCATION: (280) <223> OTHER
INFORMATION: n equals a, t, g, or c <221> NAME/KEY:
misc_feature <222> LOCATION: (288) <223> OTHER
INFORMATION: n equals a, t, g, or c <400> SEQUENCE: 7
ggcanagccg nctggtgggt gggatagacc tgctttccgg acactgagcg cctgatttag
60 ggcccttntn taggaatgcc ccanccccta ccctaagacc tattgccggg
naggattcca 120 cacttccgct cctttgggga taaacctatt aattattgct
actatcaaga gggctggggc 180 attctntgct ggtaaattcc tgaagaggca
tgactgcttt tttaagcccc aagcctctag 240 ttntgggtgt tttacggagg
ggtctnagcc tngggttgtn gtacgggngg ggttctta 298 <210> SEQ ID NO
8 <211> LENGTH: 186 <212> TYPE: DNA <213>
ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (34) <223> OTHER
INFORMATION: n equals a, t, g, or c <221> NAME/KEY:
misc_feature <222> LOCATION: (128) <223> OTHER
INFORMATION: n equals a, t, g, or c <221> NAME/KEY:
misc_feature <222> LOCATION: (155)..(156) <223> OTHER
INFORMATION: n equals a, t, g, or c <221> NAME/KEY:
misc_feature <222> LOCATION: (180) <223> OTHER
INFORMATION: n equals a, t, g, or c <400> SEQUENCE: 8
ccggcggagc aaagtccact ctgggggagc tctngcgggg accacgggcc actgctcacc
60 cactggcccc gaggggggtg tagacgccaa gactcacgca tgtttgacat
ccggagtcct 120 ggagccgngt gtcccagtgg caccactagg tgctnnctgc
ctccacagtg gggttcacan 180 ccaggg 186 <210> SEQ ID NO 9
<211> LENGTH: 308 <212> TYPE: DNA <213> ORGANISM:
Homo sapiens <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (3) <223> OTHER
INFORMATION: n equals a, t, g, or c <221> NAME/KEY:
misc_feature <222> LOCATION: (19) <223> OTHER
INFORMATION: n equals a, t, g, or c <221> NAME/KEY:
misc_feature <222> LOCATION: (24) <223> OTHER
INFORMATION: n equals a, t, g, or c <221> NAME/KEY:
misc_feature <222> LOCATION: (29) <223> OTHER
INFORMATION: n equals a, t, g, or c <221> NAME/KEY:
misc_feature <222> LOCATION: (34) <223> OTHER
INFORMATION: n equals a, t, g, or c <221> NAME/KEY:
misc_feature <222> LOCATION: (38) <223> OTHER
INFORMATION: n equals a, t, g, or c <221> NAME/KEY:
misc_feature <222> LOCATION: (40) <223> OTHER
INFORMATION: n equals a, t, g, or c <221> NAME/KEY:
misc_feature <222> LOCATION: (50) <223> OTHER
INFORMATION: n equals a, t, g, or c <221> NAME/KEY:
misc_feature <222> LOCATION: (83) <223> OTHER
INFORMATION: n equals a, t, g, or c <221> NAME/KEY:
misc_feature <222> LOCATION: (107) <223> OTHER
INFORMATION: n equals a, t, g, or c <221> NAME/KEY:
misc_feature <222> LOCATION: (205) <223> OTHER
INFORMATION: n equals a, t, g, or c <221> NAME/KEY:
misc_feature <222> LOCATION: (220) <223> OTHER
INFORMATION: n equals a, t, g, or c <221> NAME/KEY:
misc_feature <222> LOCATION: (237) <223> OTHER
INFORMATION: n equals a, t, g, or c <221> NAME/KEY:
misc_feature <222> LOCATION: (272) <223> OTHER
INFORMATION: n equals a, t, g, or c <221> NAME/KEY:
misc_feature <222> LOCATION: (242) <223> OTHER
INFORMATION: n equals a, t, g, or c <221> NAME/KEY:
misc_feature <222> LOCATION: (297) <223> OTHER
INFORMATION: n equals a, t, g, or c <221> NAME/KEY:
misc_feature <222> LOCATION: (308) <223> OTHER
INFORMATION: n equals a, t, g, or c <400> SEQUENCE: 9
ggnagaggtg acaccagcna ggtnctgtnt tggnccantn ctcccacgan ggctcaccct
60 cccctccatc tgctttaatg ctncgaatcg cctacctgtg ccctgcntgt
aaaccacagc 120 tttcaaacca gctatgggga gaggacaaca cggaggatat
tccagcttcc ccggtctggg 180 gtgaaggagt gtggggagct tgggncatcc
tcctccagtn tcctccagcc cccaggnagt 240 gnctttaanc tgtgggttgc
ccagaaaagt gncccttagg tttgttgggt tttaaangga 300 gctttaan 308
<210> SEQ ID NO 10 <211> LENGTH: 407 <212> TYPE:
DNA <213> ORGANISM: Homo sapiens <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (290)
<223> OTHER INFORMATION: n equals a, t, g, or c <221>
NAME/KEY: misc_feature <222> LOCATION: (298) <223>
OTHER INFORMATION: n equals a, t, g, or c <221> NAME/KEY:
misc_feature <222> LOCATION: (324) <223> OTHER
INFORMATION: n equals a, t, g, or c <221> NAME/KEY:
misc_feature <222> LOCATION: (326) <223> OTHER
INFORMATION: n equals a, t, g, or c <221> NAME/KEY:
misc_feature <222> LOCATION: (386) <223> OTHER
INFORMATION: n equals a, t, g, or c <221> NAME/KEY:
misc_feature <222> LOCATION: (402) <223> OTHER
INFORMATION: n equals a, t, g, or c <221> NAME/KEY:
misc_feature <222> LOCATION: (407) <223> OTHER
INFORMATION: n equals a, t, g, or c <400> SEQUENCE: 10
ggcacgagcc tgggtgtgta cggagggtct agtctgagtg cgtgtggggc ctcagaacac
60 tgtgacctta gcccagcaag ccaggccttc atgaaggcaa gaaggtgcca
ccattccctg 120 ccagcccaag actccagttc cccactgcct ctgtgtgccc
tttgcgtcct gtgaagccat 180 tgagaaatgc ccatgtgccc ctgggaaagg
gcacggctgt gtcctgacag ggtgtgtttg 240 cacagaccac caggtttcct
gtgtgtcagt attatgagga acgtcggtgn ttagagtnga 300 gcagcaggga
gttagagcag gatntntccg gggcaagtcc attttggggt tttgcggaca 360
gggcatgtta ccattgcccg aggggntaga gcagttagat tntgaan 407 <210>
SEQ ID NO 11 <211> LENGTH: 139 <212> TYPE: DNA
<213> ORGANISM: Homo sapiens <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (2) <223> OTHER
INFORMATION: n equals a, t, g, or c <221> NAME/KEY:
misc_feature <222> LOCATION: (9) <223> OTHER
INFORMATION: n equals a, t, g, or c <221> NAME/KEY:
misc_feature <222> LOCATION: (11) <223> OTHER
INFORMATION: n equals a, t, g, or c <221> NAME/KEY:
misc_feature <222> LOCATION: (45) <223> OTHER
INFORMATION: n equals a, t, g, or c <221> NAME/KEY:
misc_feature <222> LOCATION: (70) <223> OTHER
INFORMATION: n equals a, t, g, or c <221> NAME/KEY:
misc_feature <222> LOCATION: (105) <223> OTHER
INFORMATION: n equals a, t, g, or c <221> NAME/KEY:
misc_feature <222> LOCATION: (131) <223> OTHER
INFORMATION: n equals a, t, g, or c <221> NAME/KEY:
misc_feature <222> LOCATION: (137) <223> OTHER
INFORMATION: n equals a, t, g, or c <400> SEQUENCE: 11
anccagggnt ncttggtccc ccacaacctt ccccggccag gcctncagac ccagacttca
60 gccagacctn ccttaaccac caatgcagcc ggggcttgcg acaanagcag
gtgctggtct 120 tggggcagtt nttccangg 139 <210> SEQ ID NO 12
<211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 12 tcacgccata tgggctcggg gaacc
25
<210> SEQ ID NO 13 <211> LENGTH: 41 <212> TYPE:
DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 13
catccgggta ccttattaca cgaagttctc ttcctcttct g 41 <210> SEQ ID
NO 14 <211> LENGTH: 40 <212> TYPE: DNA <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 14 catcgcggat
ccgccatcat gcgtcccggg gcgccagggc 40 <210> SEQ ID NO 15
<211> LENGTH: 38 <212> TYPE: DNA <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 15 catccgggta cctcacacga
agttctcttc ctcttctg 38 <210> SEQ ID NO 16 <211> LENGTH:
40 <212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 16 catcgcggat ccgccaccat gcgtcccggg
gcgccagggc 40 <210> SEQ ID NO 17 <211> LENGTH: 35
<212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 17 tcaccgctcg agcacgaagt tctcttcctc ttctg 35
<210> SEQ ID NO 18 <211> LENGTH: 40 <212> TYPE:
DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 18
catcgcggat ccgccaccat gcgtcccggg gcgccagggc 40 <210> SEQ ID
NO 19 <211> LENGTH: 38 <212> TYPE: DNA <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 19 catccgggta
cctcacacga agttctcttc ctcttctg 38
* * * * *